Pneumococcal meningitis induces apoptosis in recently postmitotic immature neurons in the dentate gyrus of neonatal rats

Bacterial meningitis is associated with high rates of morbidity and mortality, despite advances in antibiotic therapy. Meningitis caused by Streptococcus pneumoniae is associated with a particularly high incidence of neurological sequelae including deficits resulting from damage to the hippocampus. Previous studies have documented that in neonatal rats with experimental pneumococcal meningitis, cells in the subgranular layer of the dentate gyrus undergo apoptosis. The aim of the present study was to define in more detail the nature of the dying cells in the dentate gyrus. Using bromodeoxyuridine labeling at different times before infection combined with immunocytochemistry, we identified the vulnerable cells as those which underwent mitosis 6-10 days before infection. A majority of these cells are of neuronal lineage. Thus, immature neuronal cells several days after the last cell division are preferentially triggered into apoptosis during pneumococcal meningitis. The loss of these cells may contribute to the long-lasting impairment of hippocampal function identified in animal models and in humans after bacterial meningitis.

JNK is activated but does not mediate hippocampal neuronal apoptosis in experimental neonatal pneumococcal meningitis

Pneumococcal meningitis is associated with caspase 3-dependent apoptosis of recently post-mitotic immature neurons in the dentate gyrus of the hippocampus. The death of these cells is implicated in the learning and memory deficits in patients surviving the disease. The stress-activated protein kinase c-Jun N-terminal kinase (JNK) has been shown to be an important mediator of caspase 3-dependent neuronal apoptosis. However, whether JNK is involved in hippocampal apoptosis caused by pneumococcal meningitis has so far not been investigated. Here we show in a neonatal rat model of pneumococcal meningitis that JNK3 but not JNK1 or JNK2 is activated in the hippocampus during the acute phase of infection. At the cellular level, JNK3 activation was accompanied in the dentate gyrus by markedly increased phosphorylation of its major downstream target c-Jun in early immature (Hu-positive) neurons, but not in migrating (doublecortin-positive) neurons, the cells that do undergo apoptosis. These findings suggested that JNK may not be involved in pneumococcal meningitis-induced hippocampal apoptosis. Indeed, although intracerebroventricular administration of D-JNKI-1 or AS601245 (two highly specific JNK inhibitors) inhibited c-Jun phosphorylation and protein expression in the hippocampus, hippocampal apoptosis was unaffected. Collectively, these results demonstrate that JNK does not mediate hippocampal apoptosis in pneumococcal meningitis, and that JNK may be involved in processes unrelated to apoptosis in this disease.

Expression of trefoil factor 1 in the developing and adult rat ventral mesencephalon

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides with a characteristic tree-looped trefoil structure. TFFs are mainly expressed in the gastrointestinal tract where they play a critical role in the function of the mucosal barrier. TFF1 has been suggested as a neuropeptide, but not much is known about its expression and function in the central nervous system. We investigated the expression of TFF1 in the developing and adult rat midbrain. In the adult ventral mesencephalon, TFF1-immunoreactive (-ir) cells were predominantly found in the substantia nigra pars compacta (SNc), the ventral tegmental area (VTA) and in periaqueductal areas. While around 90% of the TFF1-ir cells in the SNc co-expressed tyrosine hydroxylase (TH), only a subpopulation of the TH-ir neurons expressed TFF1. Some TFF1-ir cells in the SNc co-expressed the calcium-binding proteins calbindin or calretinin and nearly all were NeuN-ir confirming a neuronal phenotype, which was supported by lack of co-localization with the astroglial marker glial fibrillary acidic protein (GFAP). Interestingly, at postnatal (P) day 7 and P14, a significantly higher proportion of TH-ir neurons in the SNc co-expressed TFF1 as compared to P21. In contrast, the proportion of TFF1-ir cells expressing TH remained unchanged during postnatal development. Furthermore, significantly more TH-ir neurons expressed TFF1 in the SNc, compared to the VTA at all four time-points investigated. Injection of the tracer fluorogold into the striatum of adult rats resulted in retrograde labeling of several TFF1 expressing cells in the SNc showing that a significant fraction of the TFF1-ir cells were projection neurons. This was also reflected by unilateral loss of TFF1-ir cells in SNc of 6-hydroxylase-lesioned hemiparkinsonian rats. In conclusion, we show for the first time that distinct subpopulations of midbrain dopaminergic neurons express TFF1, and that this expression pattern is altered in a rat model of Parkinson's disease.

Antagonizing Nogo-receptor 1 promotes the number of cultured dopaminergic neurons and elongates their neurites

The myelin-associated protein Nogo-A and its receptor Nogo-receptor 1 (NgR1) are known as potent growth inhibitors of the adult central nervous system (CNS). Nogo-A is mostly expressed on the surface of oligodendrocytes, but is also found in neurons of the adult and developing CNS. This observation suggests that Nogo-A serves additional functions in the brain. Hence, in the present study, we investigated the effects of antagonizing NgR1 on cultured organotypic and dissociated dopaminergic neurons. For that purpose ventral mesencephalic cultures from E14 rat embryos were grown in absence or presence of the NgR1 antagonist NEP1-40 for 1 week. Treatment with NEP1-40 significantly increased cell densities of tyrosine hydroxylase-immunoreactive neurons. Moreover, organotypic ventral mesencephalic cultures displayed a significantly bigger volume after NEP1-40 treatment. Morphological analysis of tyrosine hydroxylase-positive neurons disclosed longer neurites and higher numbers of primary neurites in dissociated cultures incubated with NEP1-40, whereas soma size was not changed. In conclusion, our findings demonstrate that interfering with Nogo-A signaling by antagonizing NgR1 modulates dopaminergic neuron properties during development. These observations highlight novel aspects of the role of Nogo-A in the CNS and might have an impact in the context of Parkinson's disease.

Development of dendritic tonic GABAergic inhibition regulates excitability and plasticity in CA1 pyramidal neurons

Synaptic plasticity rules change during development: while hippocampal synapses can be potentiated by a single action potential pairing protocol in young neurons, mature neurons require burst firing to induce synaptic potentiation. An essential component for spike timing-dependent plasticity is the backpropagating action potential (BAP). BAP along the dendrites can be modulated by morphology and ion channel composition, both of which change during late postnatal development. However it is unclear whether these dendritic changes can explain the developmental changes in synaptic plasticity induction rules. Here, we show that tonic GABAergic inhibition regulates dendritic action potential backpropagation in adolescent but not pre-adolescent CA1 pyramidal neurons. These developmental changes in tonic inhibition also altered the induction threshold for spike timing-dependent plasticity in adolescent neurons. This GABAergic regulatory effect upon backpropagation is restricted to distal regions of apical dendrites (>200 μm) and mediated by α5-containing GABA(A) receptors. Direct dendritic recordings demonstrate α5-mediated tonic GABA(A) currents in adolescent neurons which can modulate backpropagating action potentials. These developmental modulations in dendritic excitability could not be explained by concurrent changes in dendritic morphology. To explain our data, model simulations propose a distally-increasing or localized distal expression of dendritic α5 tonic inhibition in mature neurons. Overall, our results demonstrate that dendritic integration and plasticity in more mature dendrites are significantly altered by tonic α5 inhibition in a dendritic region-specific and developmentally-regulated manner.

Bacterial meningitis causes two distinct forms of cellular damage in the hippocampal dentate gyrus in infant rats.

Bacterial meningitis causes neurological sequelae in up to 50% of survivors. Two pathogens known for their propensity to cause severe neurological damage are Streptococcus pneumoniae and group B streptococci. Some forms of neuronal sequelae, such as learning and memory deficits, have been associated with neuronal injury in the hippocampus. To learn more about hippocampal injury in meningitis, we performed a comparative study in bacterial meningitis due to S. pneumoniae and group B streptococcus, in which 11-day-old infant rats were infected intracisternally with either of the two pathogens. Histopathological examination of the neuronal injury in the dentate gyrus of the hippocampus showed that S. pneumoniae caused predominantly classical apoptotic cell death. Cells undergoing apoptosis were located only in the subgranular zone and stained positive for activated caspase-3 and TUNEL. Furthermore, dividing progenitor cells seemed particularly sensitive to this form of cell death. Group B streptococcus was mainly responsible for a caspase-3-independent (and TUNEL-negative) form of cell death. Compared with the morphological features found in apoptosis (e.g., apoptotic bodies), this form of neuronal death was characterized by clusters of uniformly shrunken cells. It affected the dentate gyrus throughout the blade, showing no preferences for immature or mature neurons. Thus, depending on the infecting agent, bacterial meningitis causes two distinct forms of cell injury in the dentate gyrus.

Reelin immunoreactivity in dissociated cultures of the postnatal hippocampus

Reelin is an extracellular matrix glycoprotein expressed in different nerve cell populations in the developing, early postnatal and adult central nervous system. During histogenesis of the neocortex and hippocampus, reelin is present in Cajal-Retzius cells and other early neurons and contributes to correct layering of these regions. During early postnatal life, pioneer neurons disappear and reelin expression establishes in a subpopulation of cortical and hippocampal GABAergic interneurons, where it is maintained throughout adult life. We studied the developmental distribution pattern of reelin in dissociated cultures obtained from the early postnatal hippocampus to verify whether or not such a maturation phenomenon is maintained in vitro. Reelin is expressed both in Cajal-Retzius cells and multipolar and pyramidal neurons in younger cultures. The density of reelin-positive Cajal-Retzius cells dropped drastically by about 84% in 4-week-old cultures. Multipolar and pyramidal neurons containing reelin represented 12% of the total cell population in younger cultures and decreased by about 25% after 3 to 4 weeks of cultivation. Their density was significantly lower in cultures of the same age treated with glutamate receptor antagonists. These reelin-positive multipolar and pyramidal neurons were heterogeneous, including a larger amount of non-GABAergic, and 30-40% of GABAergic neurons. Cells double labeled for reelin and the GABA synthesizing enzyme glutamic acid decarboxylase represented about 4% of the total neuron population in culture and their density remained constant with age. It is thus possible that the decrease in the total reelin population may selectively be of importance to the larger non-GABAergic fraction of reelin cells. This study shows that reelin-expressing neurons are maintained in dissociated cultures of the neonatal hippocampus and their distribution and age-dependent changes in density resemble those of the early postnatal hippocampus in vivo.

Rhythmic synaptic activity induced by mechanical injury of rat CA3 hippocampal area.

Mechanical injury of the CNS frequently results from accidents but also occurs in the course of neurosurgical interventions. A great variety of anatomical and physiological changes have been described to evolve after a brain trauma yet only little is known about processes that occur during a trauma. In the present study, I obtained whole-cell patch clamp recordings from pyramidal cells in hippocampal slice cultures while mechanically lesioning the CA3 area. Electrophysiological analysis revealed that traumatic injury massively increased excitatory and inhibitory synaptic activity in the entire CA3 region. Cutting the CA3 region induced highly rhythmic excitatory postsynaptic currents (EPSCs) that reached frequencies of around 70 Hz. Blocking voltage-dependent sodium channels with tetrodotoxin prevented the increase in synaptic activity and injury-induced neurotransmitter release in CA3 remote from the lesion site. With fast synaptic transmission blocked only neurons in the immediate vicinity of a lesion depolarized and fired action potentials upon mechanical damage. I hence suggest that mechanical injury damages the membrane and induces action potential firing in only a small population of neurons. This activity is then propagated throughout the undamaged CA3 network inducing highly rhythmic discharges. Thus mechanical brain injury initiates immediate functional changes that exceed the lesion site.

Loss of Nogo-A-expressing neurons in a rat model of Parkinson's disease

The myelin-associated protein Nogo-A is among the most potent neurite growth inhibitors in the adult CNS. Recently, Nogo-A expression was demonstrated in a number of neuronal subpopulations of the adult and developing CNS but at present, little is known about the expression of Nogo-A in the nigrostriatal system, a brain structure severely affected in Parkinson's disease (PD). The present study sought to characterize the expression pattern of Nogo-A immunoreactive (ir) cells in the adult ventral mesencephalon of control rats and in the 6-hydroxydopamine (6-OHDA) rat model of PD. Immunohistochemical analyses of normal adult rat brain showed a distinct expression of Nogo-A in the ventral mesencephalon, with the highest level in the substantia nigra pars compacta (SNc) where it co-localized with dopaminergic neurons. Analyses conducted 1week and 1 month after unilateral striatal injections of 6-OHDA disclosed a severe loss of the number of Nogo-A-ir cells in the SNc. Notably, at 1week after treatment, more dopaminergic neurons expressing Nogo-A were affected by the 6-OHDA toxicity than Nogo-A-negative dopaminergic neurons. However, at later time points more of the surviving dopaminergic neurons expressed Nogo-A. In the striatum, both small and large Nogo-A-positive cells were detected. The large cells were identified as cholinergic interneurons. Our results suggest yet unidentified functions of Nogo-A in the CNS beyond the inhibition of axonal regeneration and plasticity, and may indicate a role for Nogo-A in PD.

Effects of Forskolin on Trefoil factor 1 expression in cultured ventral mesencephalic dopaminergic neurons

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides that are mainly expressed in the gastrointestinal tract. Notably, TFF1 has been suggested to operate as a neuropeptide, however, its specific cellular expression, regulation and function remain largely unknown. We have previously shown that TFF1 is expressed in developing and adult rat ventral mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons. Here, we investigated the expression of TFF1 in rat ventral mesencephalic dopaminergic neurons (embryonic day 14) grown in culture for 5, 7 or 10 days in the absence (controls) or presence of either glial cell line-derived neurotrophic factor (GDNF), Forskolin or the combination. No TFF1-ir cells were identified at day 5 and only a few at day 7, whereas TH was markedly expressed at both time points. At day 10, several TFF1-ir cells were detected, and their numbers were significantly increased after the addition of GDNF (2.2-fold) or Forskolin (4.1-fold) compared to controls. Furthermore, the combination of GDNF and Forskolin had an additive effect and increased the number of TFF1-ir cells by 5.6-fold compared to controls. TFF1 expression was restricted to neuronal cells, and the percentage of TH/TFF1 co-expressing cells was increased to the same extent in GDNF and Forskolin-treated cultures (4-fold) as compared to controls. Interestingly, the combination of GDNF and Forskolin resulted in a significantly increased co-expression (8-fold) of TH/TFF1, which could indicate that GDNF and Forskolin targeted different subpopulations of TH/TFF1 neurons. Short-term treatment with Forskolin resulted in an increased number of TFF1-ir cells, and this effect was significantly reduced by the MEK1 inhibitor PD98059 or the protein kinase A (PKA) inhibitor H89, suggesting that Forskolin induced TFF1 expression through diverse signaling pathways. In conclusion, distinct populations of cultured dopaminergic neurons express TFF1, and their numbers can be increased by factors known to influence survival and differentiation of dopaminergic cells.