2-3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline and biocytin

Depolarization by the neurotransmitter GABA regulates adult neurogenesis. We found interneurons of the neurogliaform cell family to be a primary source of GABA for newborn neurons in mouse dentate gyrus. GABAergic depolarization occurred in concert with reduced synaptic inhibition of mature neurons, suggesting that the local circuitry coordinates the activation of new and pre-existing cells.

Topics: Action Potentials; Animals; Animals, Newborn; Bacterial Proteins; Biophysical Phenomena; Cell Adhesion Molecules, Neuronal; Electric Stimulation; Excitatory Amino Acid Antagonists; Extracellular Matrix Proteins; GABA Antagonists; Glutamic Acid; Green Fluorescent Proteins; Interneurons; Iontophoresis; Luminescent Proteins; Lysine; Mice; Mice, Inbred C57BL; Mice, Transgenic; Models, Neurological; Nerve Net; Nerve Tissue Proteins; Neural Inhibition; Neurogenesis; Neuropeptide Y; Nitric Oxide Synthase Type I; Patch-Clamp Techniques; Picrotoxin; Pro-Opiomelanocortin; Quinoxalines; Reelin Protein; Serine Endopeptidases; Stem Cell Niche; Synaptic Potentials; Time Factors; Valine

The hyperpolarization-activated cation current (I(H)) regulates the electrical activity of many excitable cells, but its precise function varies across cell types. The antiepileptic drug lamotrigine (LTG) was recently shown to enhance I(H) in hippocampal CA1 pyramidal neurons, showing a potential anticonvulsant mechanism, as I(H) can dampen dendrito-somatic propagation of excitatory postsynaptic potentials in these cells. However, I(H) is also expressed in many hippocampal interneurons that provide synaptic inhibition to CA1 pyramidal neurons, and thus, I(H) modulation may indirectly regulate the inhibitory control of principal cells by direct modulation of interneuron activity. Whether I(H) in hippocampal interneurons is sensitive to modulation by LTG, and the manner by which this may affect the synaptic inhibition of pyramidal cells has not been investigated. In this study, we examined the effects of LTG on I(H) and spontaneous firing of area CA1 stratum oriens interneurons, as well as on spontaneous inhibitory postsynaptic currents in CA1 pyramidal neurons in immature rat brain slices. LTG (100 microM) significantly increased I(H) in the majority of interneurons, and depolarized interneurons from rest, promoting spontaneous firing. LTG also caused an increase in the frequency of spontaneous (but not miniature) IPSCs in pyramidal neurons without significantly altering amplitudes or rise and decay times. These data indicate that I(H) in CA1 interneurons can be increased by LTG, similarly to I(H) in pyramidal neurons, that I(H) enhancement increases interneuron excitability, and that these effects are associated with increased basal synaptic inhibition of CA1 pyramidal neurons.

Topics: 4-Aminopyridine; Action Potentials; Animals; Animals, Newborn; Anticonvulsants; Biophysics; CA1 Region, Hippocampal; Cadmium Chloride; Calcium Channel Blockers; Electric Stimulation; Excitatory Amino Acid Antagonists; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Interneurons; Lamotrigine; Lysine; Male; Neural Inhibition; Patch-Clamp Techniques; Potassium Channel Blockers; Pyramidal Cells; Quinoxalines; Rats; Rats, Long-Evans; Tetraethylammonium; Triazines; Valine

NG2 cells, oligodendrocyte precursors, play a critical role in myelination during postnatal brain maturation, but a pool of these precursors is maintained in the adult and recruited to lesions in demyelinating diseases. NG2 cells in immature animals have recently been shown to receive synaptic inputs from neurons, and these have been assumed to persist in the adult. Here, we investigated the GABAergic synaptic activity of NG2 cells in acute slices of the barrel cortex of NG2-DsRed transgenic mice during the first postnatal month, which corresponds to the period of active myelination in the neocortex. Our data demonstrated that the frequency of spontaneous and miniature GABAergic synaptic activity of cortical NG2 cells dramatically decreases after the second postnatal week, indicating a decrease in the number of synaptic inputs onto NG2 cells during development. However, NG2 cells still receive GABAergic inputs from interneurons in the adult cortex. These inputs do not rely on the presence of functional synapses but involve a form of GABA spillover. This GABA volume transmission allows interneurons to induce phasic responses in target NG2 cells through the activation of extrasynaptic GABA(A) receptors. Hence, after development is complete, volume transmission allows NG2 cells to integrate neuronal activity patterns at frequencies occurring during in vivo sensory stimulation.

Topics: Age Factors; Animals; Animals, Newborn; Biophysics; Calcium; Cell Line, Transformed; Cerebral Cortex; Electric Conductivity; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; In Vitro Techniques; Interneurons; Luminescent Proteins; Lysine; Mice; Mice, Transgenic; Neurotransmitter Uptake Inhibitors; Nipecotic Acids; Oligodendroglia; Oximes; Patch-Clamp Techniques; Phosphinic Acids; Pyridazines; Pyridines; Quinoxalines; Statistics, Nonparametric; Stem Cells; Synapses; Synaptic Transmission

A fundamental property of neuronal networks in Ammon's horn is that each area comprises a single glutamatergic cell population and various types of GABAergic neurons. Here we describe an exception to this rule, in the form of granule cells that reside within the CA3 area and function as glutamatergic nonprincipal cells with distinct properties. CA3 granule cells in normal, healthy rats, similarly to dentate gyrus granule cells, coexpressed calbindin and the homeobox protein Prox1. However, CA3 granule cells were located outside of the dentate gyrus, often hundreds of micrometers from the hilar border, in the lucidum and radiatum layers. CA3 granule cells were present in numbers that were comparable to the rarer GABAergic neuronal subtypes, and their somato-dendritic morphology, intrinsic properties, and perforant path inputs were similar to those of dentate gyrus granule cells. CA3 granule cell axons displayed giant mossy fiber terminals with filopodial extensions, demonstrating that not all mossy fibers originate from the dentate gyrus. Somatic paired recordings revealed that CA3 granule cells innervated CA3 pyramidal and GABAergic cells similarly to conventional mossy fiber synapses. However, CA3 granule cells were distinct in the specific organization of their GABAergic inputs. They received GABAergic synapses from cholecystokinin-expressing mossy fiber-associated cells that did not innervate the dentate granule cell layer, and these synapses demonstrated unusually strong activity-dependent endocannabinoid-mediated inhibition of GABA release. These results indicate that granule cells in the CA3 constitute a glutamatergic, nonprincipal neuronal subtype that is integrated into the CA3 synaptic network.

Topics: Animals; Animals, Newborn; CA3 Region, Hippocampal; Calbindins; Cannabinoid Receptor Modulators; Cholecystokinin; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; gamma-Aminobutyric Acid; Homeodomain Proteins; In Vitro Techniques; Lysine; Membrane Potentials; Microscopy, Electron, Transmission; Nerve Net; Neurons; Neuropeptide Y; Patch-Clamp Techniques; Quinoxalines; Rats; Rats, Wistar; S100 Calcium Binding Protein G; Synapses; Tumor Suppressor Proteins

GABA, the main inhibitory neurotransmitter in the adult brain, has recently emerged as an important signal in network development. Most of the trophic functions of GABA have been attributed to depolarization of the embryonic and neonatal neurons via the activation of ionotropic GABA(A) receptors. Here we demonstrate a novel mechanism by which endogenous GABA selectively regulates the development of GABAergic synapses in the developing brain. Using whole-cell patch-clamp recordings on newborn mouse hippocampi lacking functional GABA(B) receptors (GABA(B)-Rs) and time-lapse fluorescence imaging on cultured hippocampal neurons expressing GFP-tagged brain-derived neurotrophic factor (BDNF), we found that activation of metabotropic GABA(B) receptors (GABA(B)-Rs) triggers secretion of BDNF and promotes the development of perisomatic GABAergic synapses in the newborn mouse hippocampus. Because activation of GABA(B)-Rs occurs during the characteristic ongoing physiological network-driven synaptic activity present in the developing hippocampus, our results reveal a new mechanism by which synaptic activity can modulate the development of local GABAergic synaptic connections in the developing brain.

Topics: Animals; Animals, Newborn; Brain-Derived Neurotrophic Factor; Cells, Cultured; CREB-Binding Protein; Enzyme-Linked Immunosorbent Assay; Excitatory Amino Acid Antagonists; Female; GABA Antagonists; gamma-Aminobutyric Acid; Green Fluorescent Proteins; Hippocampus; Lysine; Membrane Potentials; Mice; Mice, Knockout; Neurons; Patch-Clamp Techniques; Phosphinic Acids; Propanolamines; Quinoxalines; Receptors, GABA-B; Sodium Channel Blockers; Synapses; Tetrodotoxin; Valine

Odor coding in mammals is widely believed to involve synchronized gamma frequency (30-70 Hz) oscillations in the first processing structure, the olfactory bulb. How such inputs are read in downstream cortical structures however is not known. Here we used patch-clamp recordings in rat piriform cortex slices to examine cellular mechanisms that shape how the cortex integrates inputs from bulb mitral cells. Electrical stimulation of mitral cell axons in the lateral olfactory tract (LOT) resulted in excitation of pyramidal cells (PCs), which was followed approximately 10 ms later by inhibition that was highly reproducible between trials in its onset time. This inhibition was somatic in origin and appeared to be driven through a feedforward mechanism, wherein GABAergic interneurons were directly excited by mitral cell axons. The precise inhibition affected action potential firing in PCs in two distinct ways. First, by abruptly terminating PC excitation, it limited the PC response to each EPSP to exactly one, precisely timed action potential. In addition, inhibition limited the summation of EPSPs across time, such that PCs fired action potentials in strong preference for synchronized inputs arriving in a time window of <5 ms. Both mechanisms would help ensure that PCs respond faithfully and selectively to mitral cell inputs arriving as a synchronized gamma frequency pattern.

Topics: Action Potentials; Animals; Animals, Newborn; Bicuculline; Cerebral Cortex; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Antagonists; GABA Antagonists; gamma-Aminobutyric Acid; In Vitro Techniques; Lysine; Models, Neurological; Nerve Net; Neurons; Olfactory Pathways; Patch-Clamp Techniques; Piperidines; Quinoxalines; Rats; Rats, Sprague-Dawley; Triazines

Inflammation influences several steps of adult neurogenesis, but whether it regulates the functional integration of the new neurons is unknown. Here, we explored, using confocal microscopy and whole-cell patch-clamp recordings, whether a chronic inflammatory environment affects the morphological and electrophysiological properties of new dentate gyrus granule cells, labeled with a retroviral vector encoding green fluorescent protein. Rats were exposed to intrahippocampal injection of lipopolysaccharide, which gave rise to long-lasting microglia activation. Inflammation caused no changes in intrinsic membrane properties, location, dendritic arborization, or spine density and morphology of the new cells. Excitatory synaptic drive increased to the same extent in new and mature cells in the inflammatory environment, suggesting increased network activity in hippocampal neural circuitries of lipopolysaccharide-treated animals. In contrast, inhibitory synaptic drive was more enhanced by inflammation in the new cells. Also, larger clusters of the postsynaptic GABA(A) receptor scaffolding protein gephyrin were found on dendrites of new cells born in the inflammatory environment. We demonstrate for the first time that inflammation influences the functional integration of adult-born hippocampal neurons. Our data indicate a high degree of synaptic plasticity of the new neurons in the inflammatory environment, which enables them to respond to the increase in excitatory input with a compensatory upregulation of activity and efficacy at their afferent inhibitory synapses.

Topics: Analysis of Variance; Animals; Calcium-Binding Proteins; Dendritic Spines; Dose-Response Relationship, Radiation; Ectodysplasins; Electric Stimulation; Electroencephalography; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Green Fluorescent Proteins; Hippocampus; In Vitro Techniques; Inflammation; Inhibitory Postsynaptic Potentials; Lipopolysaccharides; Lysine; Male; Microfilament Proteins; Microscopy, Confocal; Neurogenesis; Neurons; Patch-Clamp Techniques; Quinoxalines; Rats; Rats, Sprague-Dawley; Seizures; Tetrodotoxin; Time Factors

GABA depolarizes immature cortical neurons. However, whether GABA excites immature neocortical neurons and drives network oscillations as in other brain structures remains controversial. Excitatory actions of GABA depend on three fundamental parameters: the resting membrane potential (Em), reversal potential of GABA (E(GABA)), and threshold of action potential generation (Vthr). We have shown recently that conventional invasive recording techniques provide an erroneous estimation of these parameters in immature neurons. In this study, we used noninvasive single N-methyl-d-aspartate and GABA channel recordings in rodent brain slices to measure both Em and E(GABA) in the same neuron. We show that GABA strongly depolarizes pyramidal neurons and interneurons in both deep and superficial layers of the immature neocortex (P2-P10). However, GABA generates action potentials in layer 5/6 (L5/6) but not L2/3 pyramidal cells, since L5/6 pyramidal cells have more depolarized resting potentials and more hyperpolarized Vthr. The excitatory GABA transiently drives oscillations generated by L5/6 pyramidal cells and interneurons during development (P5-P12). The NKCC1 co-transporter antagonist bumetanide strongly reduces [Cl(-)]i, GABA-induced depolarization, and network oscillations, confirming the importance of GABA signaling. Thus a strong GABA excitatory drive coupled with high intrinsic excitability of L5/6 pyramidal neurons and interneurons provide a powerful mechanism of synapse-driven oscillatory activity in the rodent neocortex in vitro. In the companion paper, we show that the excitatory GABA drives layer-specific seizures in the immature neocortex.

Topics: Age Factors; Animals; Animals, Newborn; Bumetanide; Excitatory Amino Acid Antagonists; Female; GABA Agents; gamma-Aminobutyric Acid; In Vitro Techniques; Interneurons; Lysine; Male; Membrane Potentials; Mice; Models, Neurological; Neocortex; Patch-Clamp Techniques; Pyramidal Cells; Quinoxalines; Sodium Potassium Chloride Symporter Inhibitors; Valine

Developmental changes in synaptic properties may act to limit neural and behavioral plasticity associated with sensitive periods. This study characterized synaptic maturation in a glutamatergic thalamo-cortical pathway that is necessary for vocal learning in songbirds. Lesions of the projection from medial dorsolateral nucleus of the thalamus (DLM) to the cortical nucleus lateral magnocellular nucleus of the anterior nidopallium (LMAN) greatly disrupt song behavior in juvenile birds during early stages of vocal learning. However, such lesions lose the ability to disrupt vocal behavior in normal birds at 60-70 days of age, around the time that selective auditory tuning for each bird's own song (BOS) emerges in LMAN neurons. This pattern has suggested that LMAN is involved in processing song-related information and evaluating the degree to which vocal motor output matches the tutor song to be learned. Analysis of reversed excitatory postsynaptic currents at DLM-->LMAN synapses in in vitro slice preparations revealed a pronounced N-methyl-D-aspartate receptor (NMDAR)-mediated component in both juvenile and adult cells with no developmental decrease in the relative contribution of NMDARs to synaptic transmission. However, the synaptic failure rate at DLM-->LMAN synapses in juvenile males during the sensitive period for song learning was significantly lower at depolarized potentials than at hyperpolarized potentials. In contrast, the failure rate at DLM-->LMAN synapses did not differ at hyper- versus depolarized holding potentials in adult males that had completed the acquisition of a stereotyped song. This pattern indicates that juvenile cells have a higher incidence of silent (NMDAR-only) synapses, which are postsynaptically silent at hyperpolarized potentials due to the voltage-dependent gating of NMDARs. Thus the decreased involvement of the LMAN pathway in vocal behavior is mirrored by a decline in the incidence of silent synapses but not by changes in the relative number of NMDA and AMPA receptors at DLM-->LMAN synapses. These findings suggest that a developmental decrease in silent synapses within LMAN may represent a neural correlate of behavioral plasticity during song learning.

Topics: Age Factors; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Behavior, Animal; Cerebral Cortex; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Finches; In Vitro Techniques; Learning; Lysine; Membrane Potentials; N-Methylaspartate; Nerve Net; Neural Networks, Computer; Patch-Clamp Techniques; Quinoxalines; Synapses; Thalamus; Vocalization, Animal

Paired intracellular recordings were used to investigate recurrent excitatory transmission in layers II, III and V of the rat entorhinal cortex in vitro. There was a relatively high probability of finding a recurrent connection between pairs of pyramidal neurons in both layer V (around 12%) and layer III (around 9%). In complete contrast, we have failed to find any recurrent synaptic connections between principal neurons in layer II, and this may be an important factor in the relative resistance of this layer in generating synchronized epileptiform activity. In general, recurrent excitatory postsynaptic potentials in layers III and V of the entorhinal cortex had similar properties to those recorded in other cortical areas, although the probabilities of connection are among the highest reported. Recurrent excitatory postsynaptic potentials recorded in layer V were smaller with faster rise times than those recorded in layer III. In both layers, the recurrent potentials were mediated by glutamate primarily acting at alpha-amino-3-hydroxy-5-methyl-4-isoxazole receptors, although there appeared to be a slow component mediated by N-methyl-D-aspartate receptors. In layer III, recurrent transmission failed on about 30% of presynaptic action potentials evoked at 0.2Hz. This failure rate increased markedly with increasing (2, 3Hz) frequency of activation. In layer V the failure rate at low frequency was less (19%), and although it increased at higher frequencies this effect was less pronounced than in layer III. Finally, in layer III, there was evidence for a relatively high probability of electrical coupling between pyramidal neurons. We have previously suggested that layers IV/V of the entorhinal cortex readily generate synchronized epileptiform discharges, whereas layer II is relatively resistant to seizure generation. The present demonstration that recurrent excitatory connections are widespread in layer V but not layer II could support this proposal. The relatively high degree of recurrent connections and electrical coupling between layer III cells may be a factor in it's susceptibility to neurodegeneration during chronic epileptic conditions.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Entorhinal Cortex; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; In Vitro Techniques; Lysine; Male; Neural Pathways; Pyramidal Cells; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission

Studies in slices suggest that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic currents are not present in CA1 (Cornu ammonis) pyramidal neurons at birth (P0). We have re-examined this issue in the rat intact hippocampal formation (IHF) in vitro. Injections of biocytin or carbocyanine show that the temporo-ammonic, commissural and Schaffer collateral pathways are present at birth in the marginal zone of CA1. Electrical stimulation of these pathways evoked field excitatory postsynaptic potentials (fEPSPs) in the marginal zone of CA1 from embryonic day 19 (E19) to postnatal day 9 (P9). These fEPSPs are mediated by synaptic AMPA receptors as they are reduced or completely blocked by: (i) tetrodotoxin; (ii) high divalent cation concentrations; (iii) the adenosine A1 receptor agonist CPA; (iv) anoxic episodes; (v) the selective AMPA receptor antagonist 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7, 8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine (GYKI-53655) or the mixed AMPA-kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX). The amplitude of the fEPSPs is also reduced by D(-)-2-amino-5-phosphonopentanoic acid (D-APV) and its duration is increased by bicuculline suggesting the participation of N-methyl-D-aspartate (NMDA) and GABAA (gamma-aminobutyric acid) receptors. Finally, AMPA receptor-mediated fEPSPs are also recorded in P0 slices, but they are smaller and more labile than in the IHF. Our results suggest that in embryonic CA1 neurons, glutamate acting on AMPA receptors already provides a substantial part of the excitatory drive and may play an important role in the activity-dependent development of the hippocampus. Furthermore, the IHF may be a convenient preparation to investigate the properties of the developing hippocampus.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Afferent Pathways; Aging; Animals; Animals, Newborn; Benzodiazepines; Bicuculline; Cations, Divalent; Electric Stimulation; Embryonic and Fetal Development; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Fluorescent Dyes; Hippocampus; Hypoxia; Lysine; Pyramidal Cells; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Synapses; Tetrodotoxin