Daniel Reyes-Haro

The principal neurons of the medial nucleus of the trapezoid body and NG2(+) glial cells receive coordinated excitatory synaptic input

Glial cell processes are part of the synaptic structure and sense spillover of transmitter, while some glial cells can even receive direct synaptic input. Here, we report that a defined type of glial cell in the medial nucleus of the trapezoid body (MNTB) receives excitatory glutamatergic synaptic input from the calyx of Held (CoH). This giant glutamatergic terminal forms an axosomatic synapse with a single principal neuron located in the MNTB. The NG2 glia, as postsynaptic principal neurons, establish synapse-like structures with the CoH terminal. In contrast to the principal neurons, which are known to receive excitatory as well as inhibitory inputs, the NG2 glia receive mostly, if not exclusively, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor–mediated evoked and spontaneous synaptic input. Simultaneous recordings from neurons and NG2 glia indicate that they partially receive synchronized spontaneous input. This shows that an NG2+ glial cell and a postsynaptic neuron share presynaptic terminals.

Neuron–astrocyte interactions in the medial nucleus of the trapezoid body

The calyx of Held (CoH) synapse serves as a model system to analyze basic mechanisms of synaptic transmission. Astrocyte processes are part of the synaptic structure and contact both pre- and postsynaptic membranes. In the medial nucleus of the trapezoid body (MNTB), midline stimulation evoked a current response that was not mediated by glutamate receptors or glutamate uptake, despite the fact that astrocytes express functional receptors and transporters. However, astrocytes showed spontaneous Ca2+ responses and neuronal slow inward currents (nSICs) were recorded in the postsynaptic principal neurons (PPNs) of the MNTB. These currents were correlated with astrocytic Ca2+ activity because dialysis of astrocytes with BAPTA abolished nSICs. Moreover, the frequency of these currents was increased when Ca2+ responses in astrocytes were elicited. NMDA antagonists selectively blocked nSICs while D-serine degradation significantly reduced NMDA-mediated currents. In contrast to previous studies in the hippocampus, these NMDA-mediated currents were rarely synchronized.

Zinc modulation of serotonin uptake in the adult rat corpus callosum

Antidepressants partially inhibit the uptake of 5‐hydroxytryptamine (5‐HT; serotonin) in the rat corpus callosum (CC), a white matter commissure involved in interhemispheric brain communication. It is also known that zinc modulates many proteins, including neurotransmitter transporters. We examined the effects of zinc on the uptake of 5‐HT into slices of the adult rat CC, in the absence or presence of some antidepressants. Zinc increased 5‐HT uptake in a concentration‐dependent manner when the CC slices were incubated in a solution buffered with sodium bicarbonate; however, zinc exerted no effect on 5‐HT transport when HEPES was the buffer. Potentiation of 5‐HT uptake by zinc was maximal with 1 μM (45% over the control uptake). Moreover, 1 μM zinc potentiated 5‐HT uptake in the cingulate cortex by 58% and in the Raphe nucleus by 65%. The antidepressants fluoxetine and imipramine inhibited 5‐HT uptake in the CC by ∼50%, whereas 6‐nitroquipazine, a potent 5‐HT uptake blocker, inhibited uptake by only 23%. Interestingly, inhibition of 5‐HT uptake by all three substances, fluoxetine, imipramine, and 6‐nitroquipazine, was counteracted by the presence of 1 μM zinc. Free zinc may thus contribute to modulation of extracellular levels of 5‐HT and its removal. These actions should be considered in the treatment of mental depression with antidepressants.

Expression of GABAρ receptors in the neostriatum: localization in aspiny, medium spiny neurons and GFAP-positive cells

J. Neurochem. (2012) 122, 900–910.

Regional density of glial cells in the rat corpus callosum

Axons and glial cells are the main components of white matter. The corpus callosum (CC) is the largest white matter tract in mammals; in rodents, 99% of the cells correspond to glia after postnatal day 5 (P5). The area of the CC varies through life and regional differences related to the number of axons have been previously described. Whether glial cell density varies accordingly is unknown; thus the aim of this study was to estimate glial cell density for the genu, body and splenium -the three main regions of CC-, of P6 and P30 rats. Here we report that the density of CC glial cells reduced by ~10% from P6 to P30. Even so, the density of astrocytes showed a slight increase (+6%), probably due to differentiation of glioblasts. Interestingly, glial cell density decreased for the genu (-21%) and the body (-13%), while for the splenium a minor increase (+5%) was observed. The astrocyte/glia ratio increased (from P6 to P30) for the genu (+27%), body (+17%) and splenium (+4%). Together, our results showed regional differences in glial cell density of the CC. Whether this pattern is modified in some neuropathologies remains to be explored.

Uptake of serotonin by adult rat corpus callosum is partially reduced by common antidepressants

The corpus callosum (CC) is the main white matter tract involved in interhemispheric brain communication. We establish that uptake of [3H]5‐hydroxytryptamine (5‐HT) in CC is partially inhibited by some antidepressants. Slices of the adult rat CC had a high‐affinity uptake of 5‐HT. About 80% of this uptake was Na+ dependent, with a Michaelis‐Menten constant, Km, of 420 ± 80 nM and a rate of 5‐HT uptake, Vmax, of 9.5 ± 0.8 pmol/mg protein/min. The 5‐HT uptake was reduced ∼60% at pH 5 compared with that at pH 7. Fluoxetine (Prozac) inhibited only 43% of 5‐HT uptake in a concentration‐dependent manner, with an affinity constant, Ki, of 44.7 ± 10.0 nM. We also studied the effects of other monoamine uptake inhibitors, all at 10 μM, and found that zimelidine, imipramine, and clomipramine inhibited 5‐HT uptake in the CC by ∼30–40%. The fluoxetine‐insensitive 5‐HT uptake was not altered by high concentrations of dopamine plus norepinephrine. The present data show that Na+‐dependent 5‐HT uptake occurs in the CC and optic nerve and that this uptake is partially sensitive to antidepressants and probably mediated by the serotonin transporter, which may be relevant during depression.

Dehydration-Induced Anorexia Reduces Astrocyte Density in the Rat Corpus Callosum

Anorexia nervosa is an eating disorder associated with severe weight loss as a consequence of voluntary food intake avoidance. Animal models such as dehydration-induced anorexia (DIA) mimic core features of the disorder, including voluntary reduction in food intake, which compromises the supply of energy to the brain. Glial cells, the major population of nerve cells in the central nervous system, play a crucial role in supplying energy to the neurons. The corpus callosum (CC) is the largest white matter tract in mammals, and more than 99% of the cell somata correspond to glial cells in rodents. Whether glial cell density is altered in anorexia is unknown. Thus, the aim of this study was to estimate glial cell density in the three main regions of the CC (genu, body, and splenium) in a murine model of DIA. The astrocyte density was significantly reduced (~34%) for the DIA group in the body of the CC, whereas in the genu and the splenium no significant changes were observed. DIA and forced food restriction (FFR) also reduced the ratio of astrocytes to glial cells by 57.5% and 22%, respectively, in the body of CC. Thus, we conclude that DIA reduces astrocyte density only in the body of the rat CC.

GABAρ subunits confer a bicuculline-insensitive component to GFAP+ cells of cerebellum

Significance Early postnatal development of the cerebellum involves a number of events that require signaling via the neurotransmitter GABA, which acts on specific receptors anchored in the plasma membrane. GABAergic transmission regulates the proliferation and migration of neuronal precursors of astrocytic lineage. Glial cells are known to express GABA-A receptors that include GABAρ subunits, but their expression pattern, functional properties, and trafficking dynamics remain unknown. This study found that a large number of glial cells express GABAρ in the cerebellum. Functional properties and intracellular trafficking of GABA-A receptors in glial cells grown in vitro suggest a different mechanism of GABAergic control of extrasynaptic transmission. GABA-A receptors mediating synaptic or extrasynaptic transmission are molecularly and functionally distinct, and glial cells are known to express a plethora of GABA-A subunits. Here we demonstrate that GFAP+ cells of the granular layer of cerebellum express GABAρ subunits during early postnatal development, thereby conferring peculiar pharmacologic characteristics to GABA responses. Electron microscopy revealed the presence of GABAρ in the plasma membrane of GFAP+ cells. In contrast, expression in the adult was restricted to Purkinje neurons and a subset of ependymal cells. Electrophysiological studies in vitro revealed that astrocytes express functional receptors with an EC50 of 52.2 ± 11.8 μM for GABA. The evoked currents were inhibited by bicuculline (100 μM) and TPMPA (IC50, 5.9 ± 0.6 μM), indicating the presence of a GABAρ component. Coimmunoprecipitation demonstrated protein–protein interactions between GABAρ1 and GABAα1, and double immunofluorescence showed that these subunits colocalize in the plasma membrane. Three populations of GABA-A receptors in astrocytes were identified: classic GABA-A, bicuculline-insensitive GABAρ, and GABA-A–GABAρ hybrids. Clusters of GABA-A receptors were distributed in the perinuclear space and along the processes of GFAP+ cells. Time-lapse microscopy showed GABAρ2-GFP accumulation in clusters located in the soma and along the processes. The clusters were relatively immobile, with mean displacement of 9.4 ± 0.9 μm and a net distance traveled of 1–2 μm, owing mainly to directional movement or simple diffusion. Modulation of GABAρ dynamics may be a novel mechanism of extrasynaptic transmission regulating GABAergic control of GFAP+ cells during early postnatal development.

γ-Aminobutyric acid-ρ expression in ependymal glial cells of the mouse cerebellum

The ependymal glial cells (EGCs) from the periventricular zone of the cerebellum were studied to determine their distribution and the functional properties of their γ‐aminobutyric acid type A (GABAA) receptors. EGCs were identified by the presence of ciliated structures on their ventricular surface and their expression of glial fibrillary acidic protein (GFAP). Interestingly, diverse cell types, including neurons, astrocytes, and other types of glia, were identified in the subventricular zone by their current profiles. Electron microscopy showed ciliated cells and myelinated axons in this zone, but we found no collateral connections to suggest the presence of functional synapses. GABA‐mediated currents were recorded from EGCs in cerebellar slices from postnatal days 13 to 35 (PN13–PN35). These currents were blocked by TPMPA (a highly specific GABAAρ subunit antagonist) and bicuculline (a selective antagonist for classic GABAA receptors). Pentobarbital failed to modulate GABAA‐mediated currents despite the expression of GABAα1 and GABAγ2 subunits. In situ hybridization, RT‐PCR, and immunofluorescence studies confirmed GABAρ1 expression in EGCs of the cerebellum. We conclude that cerebellar EGCs express GABAρ1, which is functionally involved in GABAA receptor‐mediated responses that are unique among glial cells of the brain.

Anorexia Reduces GFAP+ Cell Density in the Rat Hippocampus

Anorexia nervosa is an eating disorder observed primarily in young women. The neurobiology of the disorder is unknown but recently magnetic resonance imaging showed a volume reduction of the hippocampus in anorexic patients. Dehydration-induced anorexia (DIA) is a murine model that mimics core features of this disorder, including severe weight loss due to voluntary reduction in food intake. The energy supply to the brain is mediated by astrocytes, but whether their density is compromised by anorexia is unknown. Thus, the aim of this study was to estimate GFAP+ cell density in the main regions of the hippocampus (CA1, CA2, CA3, and dentate gyrus) in the DIA model. Our results showed that GFAP+ cell density was significantly reduced (~20%) in all regions of the hippocampus, except in CA1. Interestingly, DIA significantly reduced the GFAP+ cells/nuclei ratio in CA2 (−23%) and dentate gyrus (−48%). The reduction of GFAP+ cell density was in agreement with a lower expression of GFAP protein. Additionally, anorexia increased the expression of the intermediate filaments vimentin and nestin. Accordingly, anorexia increased the number of reactive astrocytes in CA2 and dentate gyrus more than twofold. We conclude that anorexia reduces the hippocampal GFAP+ cell density and increases vimentin and nestin expression.