Junichi Nabekura

Resting Microglia Directly Monitor the Functional State of Synapses In Vivo and Determine the Fate of Ischemic Terminals

Recent studies have identified the important contribution of glial cells to the plasticity of neuronal circuits. Resting microglia, the primary immune effector cells in the brain, dynamically extend and retract their processes as if actively surveying the microenvironment. However, just what is being sampled by these resting microglial processes has not been demonstrated in vivo, and the nature and function of any interactions between microglia and neuronal circuits is incompletely understood. Using in vivo two-photon imaging of fluorescent-labeled neurons and microglia, we demonstrate that the resting microglial processes make brief (∼5 min) and direct contacts with neuronal synapses at a frequency of about once per hour. These contacts are activity-dependent, being reduced in frequency by reductions in neuronal activity. After transient cerebral ischemia, the duration of these microglia–synapse contacts are markedly prolonged (∼1 h) and are frequently followed by the disappearance of the presynaptic bouton. Our results demonstrate that at least part of the dynamic motility of resting microglial processes in vivo is directed toward synapses and propose that microglia vigilantly monitor and respond to the functional status of synapses. Furthermore, the striking finding that some synapses in the ischemic areas disappear after prolonged microglial contact suggests microglia contribute to the subsequent increased turnover of synaptic connections. Further understanding of the mechanisms involved in the microglial detection of the functional state of synapses, and of their role in remodeling neuronal circuits disrupted by ischemia, may lead to novel therapies for treating brain injury that target microglia.

AMPA-kainate subtypes of glutamate receptor in rat cerebral microglia.

Microglial cells were isolated from rat cerebral cortex, and kainate (KA)-induced inward current was measured at a holding potential of −40 or −60 mV. 6-Cyano-7-nitroquinoxaline-2, 3-dione-sensitive KA-induced currents increased with increasing KA concentration. The half-activation concentration and Hill coefficient were 3.3 × 10−4m and 1.4, respectively. Although glutamate (Glu) and AMPA-induced currents were much smaller than that induced by KA, all KA-, Glu-, and AMPA-induced currents were greatly and consistently enhanced in the presence of cyclothiazide (CTZ). On the other hand, KA-induced currents were much less sensitive to potentiation by concanavain A, suggesting that the KA-induced response in rat microglia is predominantly mediated by AMPA-preferring receptors (subunits GluR1–GluR4). The current–voltage relationships of KA- and AMPA–CTZ-induced currents were almost linear or slightly outward rectifying. The reversal potential of KA-induced current shifted to negative potentials (from +4 to −40 mV) on switching from high Na+ to high Ca2+ external solution, indicating the low Ca2+ permeability through the AMPA–KA receptor channel complexes. AMPA–KA receptor expression was studied with immunohistochemistry and reverse transcription-PCR, from which GluR2, GluR3, GluR4, and GluR5 were identified. Lower levels of mRNAs for GluR7 and KA-1–KA-2 were also indicated. Finally, activation of these receptors with KA or Glu significantly enhanced the production of tumor necrosis factor-α. These results suggest that primary cultured rat microglia possesses functional Glu receptor, which may mediate neuron to microglia communication in the physiological and pathological states.

A Novel Stem Cell Source for Vasculogenesis in Ischemia: Subfraction of Side Population Cells from Dental Pulp

Cell therapy with stem cells and endothelial progenitor cells (EPCs) to stimulate vasculogenesis as a potential treatment for ischemic disease is an exciting area of research in regenerative medicine. EPCs are present in bone marrow, peripheral blood, and adipose tissue. Autologous EPCs, however, are obtained by invasive biopsy, a potentially painful procedure. An alternative approach is proposed in this investigation. Permanent and deciduous pulp tissue is easily available from teeth after extraction without ethical issues and has potential for clinical use. We isolated a highly vasculogenic subfraction of side population (SP) cells based on CD31 and CD146, from dental pulp. The CD31−;CD146− SP cells, demonstrating CD34+ and vascular endothelial growth factor‐2 (VEGFR2)/Flk1+, were similar to EPCs. These cells were distinct from the hematopoietic lineage as CD11b, CD14, and CD45 mRNA were not expressed. They showed high proliferation and migration activities and multilineage differentiation potential including vasculogenic potential. In models of mouse hind limb ischemia, local transplantation of this subfraction of SP cells resulted in successful engraftment and an increase in the blood flow including high density of capillary formation. The transplanted cells were in proximity of the newly formed vasculature and expressed several proangiogenic factors, such as VEGF‐A, G‐CSF, GM‐CSF, and MMP3. Conditioned medium from this subfraction showed the mitogenic and antiapoptotic activity on human umbilical vein endothelial cells. In conclusion, subfraction of SP cells from dental pulp is a new stem cell source for cell‐based therapy to stimulate angiogenesis/vasculogenesis during tissue regeneration.

Microglia: actively surveying and shaping neuronal circuit structure and function

The traditional role of microglia has been in brain infection and disease, phagocytosing debris and secreting factors to modify disease progression. Recent evidence extends their role to healthy brain homeostasis, including the regulation of cell death, synapse elimination, neurogenesis, and neuronal surveillance. These actions contribute to the maturation and plasticity of neural circuits that ultimately shape behavior. Here we review microglial contributions to the development, plasticity, and maintenance of neural circuits with a focus on interactions with synapses. We introduce this topic by reviewing recent studies on the migration and proliferation of microglia within the brain, and conclude with the proposal that microglia dysfunction may adversely affect brain function, and thereby contribute to the development of psychiatric and neurological disorders.

Early Changes in KCC2 Phosphorylation in Response to Neuronal Stress Result in Functional Downregulation

The K+ Cl− cotransporter KCC2 plays an important role in chloride homeostasis and in neuronal responses mediated by ionotropic GABA and glycine receptors. The expression levels of KCC2 in neurons determine whether neurotransmitter responses are inhibitory or excitatory. KCC2 expression is decreased in developing neurons, as well as in response to various models of neuronal injury and epilepsy. We investigated whether there is also direct modulation of KCC2 activity by changes in phosphorylation during such neuronal stressors. We examined tyrosine phosphorylation of KCC2 in rat hippocampal neurons under different conditions of in vitro neuronal stress and the functional consequences of changes in tyrosine phosphorylation. Oxidative stress (H2O2) and the induction of seizure activity (BDNF) and hyperexcitability (0 Mg2+) resulted in a rapid dephosphorylation of KCC2 that preceded the decreases in KCC2 protein or mRNA expression. Dephosphorylation of KCC2 is correlated with a reduction of transport activity and a decrease in [Cl−]i, as well as a reduction in KCC2 surface expression. Manipulation of KCC2 tyrosine phosphorylation resulted in altered neuronal viability in response to in vitro oxidative stress. During continued neuronal stress, a second phase of functional KCC2 downregulation occurs that corresponds to decreases in KCC2 protein expression levels. We propose that neuronal stress induces a rapid loss of tyrosine phosphorylation of KCC2 that results in translocation of the protein and functional loss of transport activity. Additional understanding of the mechanisms involved may provide means for manipulating the extent of irreversible injury resulting from different neuronal stressors.

17β-Estradiol depolarization of hypothalamic neurons is mediated by cyclic AMP

The process by which 17β-estradiol rapidly modulates the excitability of neurons in the ventromedial hypothalamus, a facilitation center of female sexual behavior and satiety center of feeding behavior, through mediation by cyclic nucleotides, was investigated by intracellular recording from the guinea pig brain slice preparations. Two types of short-term responses were produced by depolarization with decreased K+ conductance and hyperpolarization with increased K+ conductance. These two responses were enhanced by the phosphodiesterase inhibitor, isobutylmethylxanthine. However, the specific adenylate cyclase activator, forskolin, enhanced only the depolarization. The analogue of cyclic adenosine 3′,5′-monophosphate (cAMP), 8-bromo-cAMP, induced only depolarization, the ionic mechanism of which was similar to that of 17β-estradiol. In addition, the possibility of non-specific effects of cyclic nucleotides was precluded by an experiment using an analogue of cyclic guanosine 3′,5′-monophosphate (cGMP), 8-bromo-cGMP, which hyperpolarized neurons. Thus, the present study strongly suggests that the production of depolarizing responses of neurons in the hypothalamus produced by estradiol is specifically mediated through cAMP.

Neuronal Circuit Remodeling in the Contralateral Cortical Hemisphere during Functional Recovery from Cerebral Infarction

Recent advances in functional imaging of human brain activity in stroke patients, e.g., functional magnetic resonance imaging, have revealed that cortical hemisphere contralateral to the infarction plays an important role in the recovery process. However, underlying mechanisms occurring in contralateral hemisphere during functional recovery have not been elucidated. We experimentally induced a complete infarction of somatosensory cortex in right hemisphere of mice and examined the neuronal changes in contralateral (left) somatosensory cortex during recovery. Both basal and ipsilateral somatosensory stimuli-evoked neuronal activity in left (intact) hemisphere transiently increased 2 d after stroke, followed by an increase in the turnover rate of usually stable mushroom-type synaptic spines at 1 week, observed by using two-photon imaging in vivo. At 4 weeks after stroke, when functional recovery had occurred, a new pattern of electrical circuit activity in response to somatosensory stimuli was established in intact ipsilateral hemisphere. Thus, the left somatosensory cortex can compensate for the loss of the right somatosensory cortex by remodeling neuronal circuits and establishing new sensory processing. This finding could contribute to establish the effective clinical treatments targeted on the intact hemisphere for the recovery of impaired functions and to achieve better quality of life of patients.

ATP facilitates spontaneous glycinergic IPSC frequency at dissociated rat dorsal horn interneuron synapses.

1 The ATP action on spontaneous miniature glycinergic inhibitory postsynaptic currents (mIPSCs) was investigated in rat substantia gelatinosa (SG) neurons mechanically dissociated from the 2nd layer of the dorsal horn in which their presynaptic glycinergic nerve terminals remained intact. 2 ATP reversibly facilitated the frequency of the mIPSCs in a concentration‐dependent manner without affecting their amplitude distribution. The ATP agonist, 2‐methylthioATP (2MeSATP), mimicked the ATP action, while another ATP receptor agonist, αβ‐methylene‐ATP (α,β‐meATP), had no effect on mIPSCs. 3 The ATP receptor antagonists, suramin (1 × 10−6 M) and pyridoxal‐5‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (1 × 10−5 M), completely blocked the facilitatory effect of ATP on glycine release (102·0 ± 11·2 % and 99·3 ± 16·2 %, n= 6, respectively) without altering the current amplitude distributions. 4 N‐Ethylmaleimide (NEM), a sulphydryl alkylating agent, suppressed the inhibitory effect of adenosine on mIPSC frequency (111·2 ± 13·3 %, n= 4) without altering the current amplitude distribution. However, ATP still facilitated the mIPSC frequency (693·3 ± 245·2 %, n= 4) even in the presence of NEM. 5 The facilitatory effect of ATP (1 × 10−5 M) on mIPSC frequency was not affected by adding 1 × 10−4 M Cd2+ to normal external solution but was eliminated in a Ca2+‐free external solution. 6 These results suggest that ATP enhances glycine release from nerve terminals, presumably resulting in the inhibition of SG neurons which conduct nociceptive signals to the CNS. This presynaptic P2X‐type ATP receptor may function to prevent excess excitability in SG neurons, thus preventing an excessive pain signal and/or SG cell death.

Modulation of GABA A Receptor Phosphorylation and Membrane Trafficking by Phospholipase C-related Inactive Protein/Protein Phosphatase 1 and 2A Signaling Complex Underlying Brain-derived Neurotrophic Factor-dependent Regulation of GABAergic Inhibition *

Brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission, including GABAergic transmission. Exposure to BDNF alters properties of GABAA receptors and induces changes in the expression level at the cell surface. Although phospholipase C-related inactive protein-1 (PRIP-1) plays an important role in GABAA receptor trafficking and function, its role in BDNF-dependent modulation of these receptors, together with the role of PRIP-2, was investigated using neurons cultured from PRIP double knock-out mice. The BDNF-dependent inhibition of whole cell GABA-evoked currents observed in wild type neurons was not detected in neurons cultured from knock-out mice. Instead, a gradual increase in GABA-evoked currents in these neurons correlated with a gradual increase in phosphorylation of GABAA receptor β3 subunit in response to BDNF. To characterize the specific role(s) that PRIP plays as components of underlying molecular machinery, we examined the recruitment of protein phosphatase(s) to GABAA receptors. We demonstrate that PRIP associates with phosphatases as well as with β subunits. PRIP was found to colocalize with GABAA receptor clusters in cultured neurons and with recombinant GABAA receptors when co-expressed in HEK293 cells. Importantly, a peptide mimicking a domain of PRIP involved in binding to β subunits disrupted the co-localization of these proteins in HEK293 cells and potently inhibited the BDNF-mediated attenuation of GABAA receptor currents in wild type neurons. Together, the results suggest that PRIP plays an important role in BDNF-dependent regulation of GABAA receptors by mediating the specific association between β subunits of these receptors with protein phosphatases.

The effects of noradrenaline on neurones in the rat dorsal motor nucleus of the vagus, in vitro.

1. Intracellular recordings were made from vagal motoneurones identified by antidromic stimulation in the dorsal motor nucleus of the vagus (d.m.v.) in slice preparations of rat medulla oblongata. 2. Noradrenaline (NA) applied by perfusion (0.01 microM to 1 mM) depolarized 55%, hyperpolarized 32% and produced a biphasic response (hyperpolarization followed by depolarization) in 9% of the d.m.v. neurones tested. 3. The NA effects persisted after complete elimination of synaptic inputs during perfusion with Ca2+‐free high‐Mg2+ solution, and therefore probably resulted from a direct action on the postsynaptic membranes. 4. The NA depolarization was blocked by prazosin and the NA hyperpolarization by yohimbine, but neither was blocked by propranolol or timolol. Phenoxybenzamine blocked both responses. The results indicate that NA depolarization is mediated by alpha 1‐adrenoceptors and hyperpolarization by alpha 2‐adrenoceptors. 5. The neurones which were depolarized by NA were also hyperpolarized by NA when the alpha 1‐adrenoceptors were blocked by prazosin (all of seven neurones tested). This result suggests that most vagal motoneurones in the d.m.v. have both alpha 1‐and alpha 2‐adrenoceptors. 6. The NA depolarization was accompanied by a decrease in membrane conductance and the hyperpolarization by an increase in membrane conductance, both of which were measured under manual‐clamp conditions. 7. The reversal potentials for the NA responses were around ‐85 mV in normal Ringer solution, and shifted as predicted by the Nernst equation when the extracellular K+ concentration was changed. 8. The inhibitory postsynaptic potentials evoked by focal electrical stimulation on the slice surface of the commissural part of the nucleus of the tractus solitarius (n.t.s.), which contains an A2 catecholaminergic cell group, were abolished by yohimbine. 9. The results suggest that NA modulates vagal output by decreasing or increasing the K+ conductance of d.m.v. neurones through alpha 1‐ or alpha 2‐adrenoceptors. In addition, the A2 noradrenergic cell group within the n.t.s. may send inhibitory inputs to the d.m.v.