Donna L. Gruol

Physiological and pathological roles of interleukin-6 in the central nervous system

The cytokine interleukin-6 (IL-6) is an important mediator of inflammatory and immune responses in the periphery. IL-6 is produced in the periphery and acts systemically to induce growth and differentiation of cells in the immune and hematopoietic systems and to induce and coordinate the different elements of the acute-phase response. In addition to these peripheral actions, recent studies indicate that IL-6 is also produced within the central nervous system (CNS) and may play an important role in a variety of CNS functions such as cell-to-cell signaling, coordination of neuroimmune responses, protection of neurons from insult, as well as neuronal differentiation, growth, and survival. IL-6 may also contribute to the etiology of neuropathological disorders. Elevated levels of IL-6 in the CNS are found in several neurological disorders including AIDS dementia complex, Alzheimers disease, multiple sclerosis, systemic lupus erythematosus, CNS trauma, and viral and bacterial meningitis. Moreover, several studies have shown that chronic overexpression of IL-6 in transgenic mice can lead to significant neuroanatomical and neurophysiological changes in the CNS similar to that commonly observed in various neurological diseases. Thus, it appears that IL-6 may play a role in both physiological and pathophysiological processes in the CNS.

ASSOCIATION BETWEEN THE NUCLEOLUS AND THE COILED BODY

By means of light and electron microscopic immunocytochemistry, we have localized p80-coilin, a specific protein marker for coiled bodies, in mammalian cell lines as well as in primary rat neuron cultures. p80-coilin-stained nuclear bodies, which also contained fibrillarin, could be subsequently silver stained by a method specific for the visualization of nucleolar organizer regions. In cycling cells, most coiled bodies were not associated with nucleoli, whereas in rat neurons such as association was frequent. The treatment of cycling cells with actinomycin D or 5,6-dichloro-1-beta-D-ribo furanosyl-benzimidazole led to nucleolar segregation and/or disintegration, and to an association of p80-coilin staining structures with nucleoli. p80-coilin-positive structures contained fibrillarin in both untreated and treated cells. These results support the opinion that there might be a special association between coiled bodies and nucleoli, particularly in neuronal cells.

Neuronal Expression of a Functional Receptor for the C5a Complement Activation Fragment

The present studies were undertaken to determine whether neuronal subsets in normal brains constitutively express functionally competent C5a receptors. In situ hybridization studies coupled with immunohistochemical approaches revealed that most neurons in the hippocampal formation, many pyramidal cortical neurons, and cerebellar Purkinje neurons in normal human and murine brains constitutively express C5a receptors. Neuronal C5a receptors bound C5a-coated fluorescent microspheres, and primary rodent hippocampal neurons responded to C5a with increased calcium fluxes via a pertussis-sensitive, presumably Gi-coupled protein. Additional studies with human neuroblastoma cells conducted to address the functional role of C5a receptors revealed that C5a triggered rapid activation of protein kinase C and activation and nuclear translocation of the NF-κB transcription factor. In addition, C5a was found to be mitogenic for undifferentiated human neuroblastoma cells, a novel action for the C5aR. In contrast, C5a protected terminally differentiated human neuroblastoma cells from toxicity mediated by the amyloid Aβ peptide. Thus, normal rodent hippocampal neurons as well as undifferentiated and differentiated human neuroblastoma cells express functional C5a receptors. These results have implications for understanding the role of neuronal C5aR receptors in normal neuronal development, neuronal homeostasis, and neuroinflammatory conditions such as Alzheimer’s disease.

Handbook of the cerebellum and cerebellar disorders

The cerebellar primordium develops dorsally at an intermediate anteroposterior (AP) level of the neural tube. Its size is modulated by the early anteriorizing and posteriorizing signals, which pattern the neural tube. Two important signaling centers, the midbrain–hindbrain organizer and the roof plate, intersect at the level of the cerebellar anlage and control its positioning, differentiation, growth, survival, and patterning. Neural tube bending in the pontine region induces a widening of the fourth ventricle, which is made possible by choroid plexus differentiation and extension. As a consequence of these morphogenetic changes, the AP axis of the cerebellar primordium is rotated by 90 , and the cerebellar vermis and hemispheres derive from the anterior and posterior parts of the early cerebellar plate, respectively. The cerebellar plate is progressively subdivided along its dorsoventral axis into distinct domains, which generate subsets of cerebellar neurons according to their neurotransmitter phenotype. The roof plate marked by Gdf7 expression is at the origin of choroid plexus cells but does not contribute neurons or glia to the cerebellum. The rhombic lip, marked by Atoh1 expression, produces all the glutamatergic neurons of the cerebellum and a large number of non-cerebellar neurons. Finally, the ventral cerebellar neuroepithelium, marked by Ptf1a expression, generates all the GABAergic neurons and can be further subdivided into two progenitor domains, devoted to the production of Purkinje cells and GABAergic projection neurons of the deep M. Wassef Institut de Biologie de l’Ecole Normale Supérieure (IBENS), 46 rue d’Ulm, 75005 Paris, France and CNRS UMR 8197, 46 rue d’Ulm, 75005 Paris, France and INSERM U1024, 46 rue d’Ulm, 75005 Paris, France e-mail: wassef@biologie.ens.fr M. Manto, D.L. Gruol, J.D. Schmahmann, N. Koibuchi, F. Rossi (eds.), Handbook of the Cerebellum and Cerebellar Disorders, DOI 10.1007/978-94-007-1333-8_1, # Springer Science+Business Media Dordrecht 2013 3 cerebellar nuclei. The so-called cerebellar primordium is not restricted to the production of cerebellar neurons but contributes to a large number of nuclei in the isthmic region.

Cultured cerebellar neurons: endogenous and exogenous components of Purkinje cell activity and membrane response to putative transmitters.

Modified explant cultures of fetal rat cerebellum were developed for electrophysiological and pharmacological studies, at the membrane level, of Purkinje neurons. The goals of the present series of experiments were to identify possible endogenous and exogenous components to the electrical activity of Purkinje neurons, to assess the sensitivity of these neurons to putative excitatory and inhibitory neurotransmitters, and to characterize the membrane response to the transmitters. Intracellular recordings were made from Purkinje neurons, identified on a morphological basis, using conventional electrophysiological techniques. Virtually all Purkinje neurons displayed spontaneous activity. A contribution of both endogenous and exogenous components to the spontaneous activity was indicated by alterations in the pattern and amount of activity when the membrane potential was varied and by the characteristics of the individual potentials themselves. Several types of activity were considered to be endogenous: the most common type consisted of pacemaker-like potentials which generated a pattern of firing similar to that characterized as simple spike activity in previous in vivo studies; another type of endogenous activity consisted of large membrane depolarizations that evoked one or two spikes. These depolarizing responses were similar to the membrane response generated by climbing fiber input to Purkinje cells in vivo. The exogenous components to the spontaneous activity consisted of synaptic potentials including excitatory (EPSPs) and inhibitory (IPSPs) synaptic potentials and biphasic EPSP/IPSPs. Several putative transmitters thought to mediate these synaptic potentials were tested by focal micropressure application to determine if they could mimic the action of the endogenous transmitters. The putative transmitter glutamate depolarized the cultured Purkinje neurons and evoked action potentials, characteristics which were displayed by the excitatory synaptic potentials. The putative inhibitory transmitter GABA hyperpolarized the cultured Purkinje neurons and depressed activity, characteristics which were displayed by the inhibitory synaptic potentials. The putative inhibitory transmitters glycine and taurine were ineffective. Norepinephrine, the transmitter mediating the inhibitory input from the locus coeruleus to Purkinje neurons, was also tested. When applied in the microM range, NE effects were variable. When applied in the mM range, NE depressed the spontaneous activity in a manner suggestive of a presynaptic action.

Corticotropin-releasing factor suppresses the afterhyperpolarization in cerebellar Purkinje neurons

Corticotropin-releasing factor (CRF) is present in climbing fibers and their terminations on Purkinje neurons. To determine whether CRF may have a postsynaptic action in this pathway, CRF effects on the electrophysiological properties of cultured cerebellar Purkinje neurons were studied. CRF produced a dose-dependent reduction of the amplitude of the afterhyperpolarization (AHP) which follows a current-induced spike train. The effect of CRF on the AHP was specific in that CRF did not significantly alter input resistance, membrane potential, amplitude of the depolarizing-off response produced at the termination of a hyperpolarizing current pulse, or the number of depolarization-induced simple spikes. Suppression of the AHP by CRF may be one mechanism by which CRF regulates the response of Purkinje neurons to other transmitters in the climbing fiber pathway or in other inputs to Purkinje neurons.

Interleukin-6, β-amyloid peptide and NMDA interactions in rat cortical neurons

Neuronal damage in Alzheimers disease (AD) is thought to involve direct toxicity of β-amyloid peptide (Aβ) and excitotoxicity involving NMDA receptors (NMDARs) and altered Ca2+ dynamics. Inflammation agents produced by microglia or astrocytes and associated with senile plaques such as the cytokine interleukin-6 (IL-6) could also contribute. To investigate this possibility, neuronal damage (lactate dehydrogenase assay, LDH, assay) was measured in cultures of rodent cortical neurons chronically treated with IL-6, Aβ or Aβ plus IL-6 and acutely treated with NMDA. Both Aβ and NMDA produced neuronal damage and this effect was larger with combined treatment. IL-6 did not produce significant neuronal damage but the largest neuronal damage was observed in cultures exposed to all three factors. IL-6 and Aβ enhanced Ca2+ responses to NMDA and combined treatment produced the largest effect. These results are consistent with a role for interactions between Aβ, NMDA and IL-6 in the neuronal loss in AD.

Two pharmacologically distinct histamine receptors mediating membrane hyperpolarization on identified neurons of Aplysia californica

Two distinct hyperpolarizing responses are produced when histamine is iontophoretically applied onto the somal membranes of identified neurons within the cerebral ganglion of Aplysia: a biphasic response consisting of a rapid component (less than 5 sec) usually superimposed upon a slowly developing component; or a monophasic slowly developing response 5-20 sec in duration. The reversal potential values for the fast (typically -65 mV) and the slow (typically -89 mV) responses, and their shift to new values when the external potassium or chloride concentrations were altered, revealed that the fast and slow potentials are produced predominantly by conductance increases to chloride and potassium ions, respectively. The effects of histamine H1- and H2-receptor agonists and antagonists were studied to characterize the pharmacological properties of histamine receptors mediating these two ionically dissimilar hyperpolarizing responses. The slow potassium-dependent hyperpolarization could be mimicked by several histamine analogues; the most potent tested were the H1-receptor agonist, 2-methylhistamine, and the H2-receptor agonist, 4-methylhistamine. Neither of these agents mimicked the fast chloride-dependent histamine response. The slow potassium-dependent responses induced by histamine or histamine agonists were completely and reversibly blocked by the H2-receptor antagonist, cimetidine. By contrast, the slow potassium-dependent hyperpolarizations produced by iontophoretically applied acetylcholine or by dopamine to the same neurons were unaffected by cimetidine. Other H1 and H2 antagonists tested were either ineffective, or only partially blocked the slow hyperpolarizations in a non-selective manner. The fast chloride-dependent hyperpolarizations were not selectively antagonized by any of the H1 or H2 reagents tested, although they were effectively suppressed by tubocurarine and strychnine. These data indicate that two pharmacologically distinct histamine receptors mediate potassium- and chloride-dependent hyperpolarizations in Aplysia neurons. Neither of these receptors, however, could be classified as strictly H1 or H2 according to criteria presently used in non-neuronal tissues. The selectivity and reversibility of cimetidine indicate that this particular antihistaminic could be a valuable pharmacological tool for defining putative histaminergic synapses in Aplysia and perhaps other nervous systems.

Dehydroepiandrosterone sulfate alters synaptic potentials in area CA1 of the hippocampal slice

The influences of the neurosteroid dehydroepiandrosterone sulfate (DHEAS) on neuronal properties and synaptic transmission in area CA1 of the hippocampus were examined using a slice preparation. DHEAS had no apparent effects upon cell membrane resistance or active cell responses to intracellular hyperpolarizing or depolarizing current pulses. However, DHEAS did increase the excitability of CA1 neurons in response to Schaffer collateral synaptic stimulation. This was apparent both in field potential recordings as well as intracellular recordings. Effects appeared within minutes following exposure to DHEAS and were reversible, suggesting a non-genomic mechanism of action. Intracellular recordings indicated that DHEAS increased the amplitudes of EPSPs associated with stimulation of Schaffer collateral fibers. The increased EPSP amplitudes resulted from DHEAS effecting an inhibition of fast IPSPs as well as a direct enhancement of excitatory synaptic transmission. No significant effects on slow IPSPs were noted. In summary, neurosteroids such as DHEAS may influence synaptic transmission through multiple mechanisms. Such influences result in increased excitability of postsynaptic neurons and indicate the profound influences neurosteroids may have to regulate neuronal activity in intact CNS structures.

Chronic intermittent ethanol exposure enhances NMDA-receptor-mediated synaptic responses and NMDA receptor expression in hippocampal CA1 region

In previous studies, we found that chronic intermittent ethanol (CIE) treatment-a model of ethanol consumption in which animals are exposed to and withdrawn from intoxicating levels of ethanol on a daily basis-produces neuroadaptive changes in hippocampal area CA1 excitatory synaptic transmission and plasticity. Synaptic responses mediated by N-methyl-D-aspartate (NMDA) receptors are known to be sensitive to ethanol and could play an important role in the neuroadaptive changes induced by CIE treatment. To address this issue, we compared electrophysiological recordings of pharmacologically isolated NMDA-receptor-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 region of hippocampal slices prepared from control rats and rats exposed to 2 weeks of CIE treatment administered by vapor inhalation. We found that fEPSPs induced by NMDA receptor activation were unaltered in slices prepared shortly after cessation of CIE treatment (i.e., < or = 1 day of withdrawal from CIE). However, following 7 days of withdrawal from CIE treatment, NMDA-receptor-mediated fEPSPs were augmented relative to age-matched controls. Western blot analysis of NMDA receptor subunit expression showed that, at 7 days of withdrawal, the level of protein for NR2A and NR2B subunits was elevated in the CA1 region of hippocampal slices from CIE-treated animals compared with slices from age-matched controls. These results are consistent with an involvement of NMDA-receptor-mediated synaptic responses in the neuroadaptive effects of CIE on hippocampal physiology and suggest that such changes may contribute to ethanol-induced changes in processes dependent on NMDA-receptor-mediated synaptic responses such as learning and memory, neural development, hyperexcitability and seizures, and neurotoxicity.