Cell Body Layer Research Articles

Structural bases of intracortical processes underlying the synchronization of epileptic potentials in the sensorimotor areas of the neocortex in rats.

Studies in long-term isolated areas of the rat neocortex were performed to investigate the dynamics of the numbers and areas of nerve cell bodies in layer V and to compare these data with the degree of synchronization of epileptic discharges evoked by application of penicillin. Decreases in the number of pyramidal neurons with body areas of 200-350 microm2 in isolated strips after maintenance for 30 and 90 days led to decreases in the degree of synchronization of epileptiform potentials. Large pyramidal neurons are known to have long horizontal axon collaterals, spreading into layers V and VI of the neocortex. It is suggested that the neural networks formed by large pyramidal neurons by means of their long horizontal collaterals mediate the process of intracortical synchronization.

Optical recording of responses to odor in olfactory structures of the nervous system in the terrestrial mollusk Helix.

Electrophysiological methods and optical methods based on the use of potential-sensitive dyes were used to record stable rhythmic oscillations of local potentials in the olfactory structure (procerebrum) of the pulmonate mollusk Helix: these oscillations were generally similar to those previously observed in slugs. Odor had the effect of transiently altering rhythmic oscillations to generate an individual pattern. This is the first study describing the recording of procerebrum potentials evoked by presentation of odor, with mapping of the areas of propagation of these potentials relative to the areas of propagation of rhythmic oscillations. The boundary of the propagation of the evoked potential was essentially similar to the projection of the neuropil, and rhythmic oscillations were recorded in the projection layer of procerebrum cell bodies. Evoked potential waves appeared in areas corresponding to the site at which the olfactory nerve enters the cerebral ganglion (of which the procerebrum forms a part) and were propagated in the procerebrum neuropil towards the cell body layer. Evoked potentials did not provoke out-of-phase waves of rhythmic oscillations.

Astrocytic clasmatodendrosis in hippocampal organ culture.

Mechanisms by which astrocytes are irreversibly injured from ischemic brain injury remain incompletely defined. More than 90 years ago Alzheimer showed that astrocytes lose their distal processes (i.e., undergo "clasmatodendrosis") when irreversibly injured by a reduction in blood flow, a process shown by Friede and van Houten (1961) to be due to energy failure and acidosis. Such alterations in astrocytic morphology can relate directly to changes in cell function. However, astrocytic clasmatodendrosis has largely been lost to the modern literature, perhaps because of a inability to study it under controlled conditions. In the present study, novel four-dimensional (4D)and digital deblurring imaging of glial fibrillary acidic protein (GFAP) immunostaining changes in hippocampal organ cultures (HOTCs) were used to establish an in vitro model of astrocytic clasmatodendrosis. Also, astrocytes in primary culture were transfected with green fluorescent protein (GFP) to show the occurrence of clasmatodendrosis via a parallel and separate means. In HOTCs, a significant reduction in astrocytic process length occurred 15 min (and remained for 60 min) after exposure to acidic Ringer's and mitochondrial inhibition in the pyramidal cell body layer. Time-lapsed images of primary cultures showed thinning of cell processes within 15 min of exposure to acidic Ringer's and mitochondrial inhibition. Distal processes subsequently broke away but retained their fluorescence for minutes before disintegrating along with their parent cell bodies. This report shows the spatiotemporal occurrence of clasmatodendrosis in astrocytes of HOTCs closely parallels that seen in vivo. Thus, HOTCs, where microenvironmental conditions can be controlled and single, identified cells can be followed in space and time, can be applied to study the interrelations between energy metabolism and pH that result in clasmatodendrosis.

Origins and Distribution of Cholinergically Induced β Rhythms in Hippocampal Slices

Regional variations and substrates of high-frequency rhythmic activity induced by cholinergic stimulation were studied in hippocampal slices with 64-electrode recording arrays. (1) Carbachol triggered beta waves (17.6 +/- 5.7 Hz) in pyramidal regions of 75% of the slices. (2) The waves had phase shifts across the cell body layers and were substantially larger in the apical dendrites than in cell body layers or basal dendrites. (3) Continuous, two-dimensional current source density analyses indicated apical sinks associated with basal sources, lasting approximately 10 msec, followed by apical sources and basal sinks, lasting approximately 20 msec, in a repeating pattern with a period in the range of 15-25 Hz. (4) Carbachol-induced beta waves in the hippocampus were accompanied by 40 Hz (gamma) oscillations in deep layers of the entorhinal cortex. (5) Cholinergically elicited beta and gamma rhythms were eliminated by antagonists of either AMPA or GABA receptors. Benzodiazepines markedly enhanced beta activity and sometimes introduced a distinct gamma frequency peak. (6) Twenty Hertz activity after orthodromic activation of field CA3 was distributed in the same manner as carbachol-induced beta waves and was generated by a current source in the apical dendrites of CA3. This source was eliminated by high concentrations of GABA(A) receptor blockers. It is concluded that cholinergically driven beta rhythms arise independently in hippocampal subfields from oscillatory circuits involving (1) bursts of pyramidal cell discharges, (2) activation of a subset of feedback interneurons that project apically, and (3) production of a GABA(A)-mediated hyperpolarization in the outer portions of the apical dendrites of pyramidal neurons.

Optical recording of odor-evoked responses in the olfactory brain of the naïve and aversively trained terrestrial snails.

Regular spontaneous oscillations were recorded both electro- and optophysiologically using a voltage-sensitive absorption dye in the olfactory part of the brain (procerebral lobe of the cerebral ganglia) of the gastropod mollusk Helix lucorum. Odor application caused transient changes in procerebral oscillations, and an odor-evoked potential was recorded in the procerebrum (PC). The wave of evoked potential originated near the place of olfactory nerve entrance into the PC and propagated via the procerebral neuropile toward the cell body layer. The spread of the odor-evoked potential corresponded roughly to the neuropile area, whereas the spontaneous oscillations were recorded in the cell body layer of the PC and were not observed in the neuropile. Evoked potential did not produce additional events intercalated into the ongoing spontaneous oscillations. Changes in parameters of spontaneous oscillations to the repeated presentations of the same odor were variable. To estimate the role of spontaneous oscillations in odor encoding, we trained the snail to avoid cineole, using paired presentations of cineole and electric shock. Elaboration of conditioned aversion to cineole applications resulted in distinct pairing-specific changes in behavior of the snails and procerebral activity. Responses to odor (cineole) applications were not different in amplitude or frequency of spontaneous oscillations in control and trained snails, whereas ratio of amplitudes of the same oscillation wave in proximal and distal regions of the procerebrum was significantly different in control and aversively trained snails, reflecting changes in neural firing in certain areas of the olfactory lobe.

Zinc-rich synaptic boutons in human temporal cortex biopsies

The distribution of zinc-rich synaptic boutons in biopsies of the temporal cortex from epileptic patients who had undergone surgery is described. Unfixed cryostat sections were exposed to H 2S vapour to precipitate endogenous zinc, which was subsequently shown by silver enhancement. In the temporal cortex, the stain for zinc was arranged in bands: stain was heavy in layers II and VI, moderate-to-heavy in layers I, III and V, and low in layer IV. The white matter was virtually devoid of staining. At the electron microscope level, labelling was found in synaptic boutons that made asymmetric synaptic contacts. Immunohistochemical staining for glutamate receptor subunits GluR2/3 was observed in cell bodies in layers II, III, V and VI, coincident with the layers that showed heavy staining for zinc. Immunostaining for glutamate receptor subunit GluR1 was prominent in non-pyramidal neurons in deep cortical layers. These results support findings in other mammals and indicate that the human neocortex may contain an extensive system of zinc-rich cortico-cortical connections. This system may be altered in pathological conditions.

Neural activity in the horizontal limb of the diagonal band of broca can be modulated by electrical stimulation of the olfactory bulb and cortex in rats.

Previously published theoretical models of olfactory processing suggest that cholinergic modulatory inputs to the olfactory system should be regulated by neural activity in the olfactory bulb. We tested these predictions using in vivo electrophysiology in rats. We show that the activity of approximately 20% of neurons recorded in the horizontal limb of the diagonal band of Broca (HDB), which is the source of cholinergic projections to the olfactory system, can be modulated by electrical stimulation of either the lateral olfactory tract or the cell body layer of piriform cortex. These data suggest a possible physiological pathway for the proposed regulation of neural activity in the HDB by activity in the olfactory bulb or cortex.

Neurotrophin receptors in the somatosensory cortex of the mature rat: co-localization of p75, trk, isoforms and c- neu

Trk immunoreactivity is expressed by a discrete population of cortical neurons, primarily those with cell bodies in layer Vb and dendrites in supragranular cortex. We tested the hypothesis that neurons co-express multiple isoforms of trk receptors. The distribution of neurons expressing specific high affinity neurotrophin receptors was determined immunohistochemically. Multiple antibodies directed against each trk isoform and an antibody directed against an epitope shared by all three trk isoforms were used. The distribution of neurons expressing each of the three receptors was virtually identical. Each anti- trk antibody primarily labeled neurons with cell bodies in layer V. More than one-third of layer V neurons was positive for a high affinity trk receptor. Few immunoreactive somata (1%–5%) were in the other layers. In addition, the neuropil in the supragranular laminae was immunopositive for each trk isoform. Recent data show that layer V neurons in the mature somatosensory cortex express the tyrosine kinase receptor c- erbB2, also known as c- neu. Immunofluorescence double labeling shows that ∼80% of the c- neu-immunolabeled neurons in layer V co-expressed pan- trk immunoreactivity and two-thirds of all c- neu-positive neurons expressed a specific trk isoform. We concluded from these data that there is significant co-expression of trk isoforms in layer V neurons. In summary, trkA, trkB, trkC, and c- neu were primarily expressed by cortical projection neurons in layer V and co-expression among these receptors was common. This implies that cortical growth factor systems are redundant and that cortical neurons are responsive to more than one growth factor.

Expression of nerve growth factor and its receptors in the somatosensory-motor cortex of Macaca nemestrina.

The present study was designed to examine the nerve growth factor (NGF) system (ligand and receptor-expressing neurons) in the somatosensory (areas 1, 3a, and 3b) and motor (area 4) cortices of the mature macaque. Light and electron microscope immunohistochemistry was used to assess the distribution and identity of NGF-, p75-, and trk-expressing elements. In each cortical area examined, NGF-positive neuronal somata were distributed through all laminae; most immunolabeled neurons were in layers II, III, and V. Based upon light microscope criteria (e.g., the morphology of proximal dendrites), both pyramidal and stellate neurons expressed NGF. Of the identifiable NGF- immunoreactive cells, 92% were pyramidal neurons and the remainder was stellate neurons. The electron microscope study showed that most (88%) NGF-positive somata formed symmetric synapses, whereas the others formed both symmetric and asymmetric synapses. As the somata of pyramidal neurons form only symmetric synapses and those of inhibitory stellate neurons form both symmetric and asymmetric somatic synapses, the ultrastructural data support the light microscopic analyses. In contrast, neurotrophin receptors, p75 and trk, were expressed chiefly by the cell bodies of layer V pyramidal neurons and the supragranular neuropil. At the ultrastructural level, receptor-positive profiles were post-synaptic elements (e.g., dendritic shafts and spines) and the concentration of immunoreactivity was greatest in the vicinity of post-synaptic densities. Thus, NGF regulatory systems parallel excitatory and inhibitory neurotransmitter systems. Cortex contains the morphological framework by which pyramidal and/or inhibitory stellate neurons can affect the activity of post-synaptic pyramidal neurons via anterograde and autocrine/paracrine NGF systems.

Quantitative assessment of localization and colocalization of glutamate, aspartate, glycine, and GABA immunoreactivity in the chick retina.

We examined the posthatch chick retina for the frequency of occurrence of localization and colocalization of four amino acid transmitter candidates: glutamate (Glu), aspartate (Asp), gamma aminobutyric acid (GABA), and glycine (Gly) using postembedding methods. We support previous studies of Glu, Asp, GABA, and Gly localization in the direct and indirect functional pathways of the chick retina and extend these studies with new qualitative and quantitative observations. We found that photoreceptors show distinct cellular immunoreactivity for both Glu (Glu+) and Asp+, but not for Gly (Gly-) or GABA. Moreover, there is compartmentalization of Glu and Asp staining within the photoreceptors. All horizontal cells react strongly with Asp and Glu, about three-fourths are GABA+ and three-fourths of these are Gly+. Bipolar cells are uniformly Glu+, heterogeneously Asp+, occasionally Gly+, but GABA-. A majority of amacrine cells stain heterogeneously with all antibodies: 90% are Gly+, slightly more than half colocalize Glu, GABA, and Gly. Furthermore, amacrine cells in the outer two or three rows of cells are more likely to be stained by Gly than Glu, Asp, or GABA. Confirming previous studies, ganglion cells were mostly immunoreactive for Glu and Asp with fewer reactive for GABA and Gly. Strong and distinctly cellular immunoreactivity was found in both central and peripheral retina. Our findings show: 1) there is extensive colocalization of Glu, Asp, GABA, and Gly among most retinal neurons, including some cells that contain all four; 2) cells of the direct functional pathway tend to be labeled by Glu and Asp generally to the exclusion of GABA and Gly, while those of the indirect pathway tend to be labeled by GABA+ and/or Gly+ in addition to Glu+ and Asp+; 3) different cell body layers have distinct patterns of colocalization; and 4) there is no qualitative difference in staining patterns between peripheral and central retina.

Chloride-cotransport blockade desynchronizes neuronal discharge in the "epileptic" hippocampal slice.

Antagonism of the chloride-cotransport system in hippocampal slices has been shown to block spontaneous epileptiform (i.e., hypersynchronized) discharges without diminishing excitatory synaptic transmission. Here we test the hypotheses that chloride-cotransport blockade, with furosemide or low-chloride (low-[Cl(-)](o)) medium, desynchronizes the firing activity of neuronal populations and that this desynchronization is mediated through nonsynaptic mechanisms. Spontaneous epileptiform discharges were recorded from the CA1 and CA3 cell body layers of hippocampal slices. Treatment with low-[Cl(-)](o) medium led to cessation of spontaneous synchronized bursting in CA1 >/=5-10 min before its disappearance from CA3. During the time that CA3 continued to burst spontaneously but CA1 was silent, electrical stimulation of the Schaffer collaterals showed that hyperexcited CA1 synaptic responses were maintained. Paired intracellular recordings from CA1 pyramidal cells showed that during low-[Cl(-)](o) treatment, the timing of action potential discharges became desynchronized; desynchronization was identified with phase lags in firing times of action potentials between pairs of neurons as well as a with a broadening and diminution of the CA1 field amplitude. Continued exposure to low-[Cl(-)](o) medium increased the degree of the firing-time phase shifts between pairs of CA1 pyramidal cells until the epileptiform CA1 field potential was abolished completely. Intracellular recordings during 4-aminopyridine (4-AP) treatment showed that prolonged low-[Cl(-)](o) exposure did not diminish the frequency or amplitude of spontaneous postsynaptic potentials. CA3 antidromic responses to Schaffer collateral stimulation were not significantly affected by prolonged low-[Cl(-)](o) exposure. In contrast to CA1, paired intracellular recordings from CA3 pyramidal cells showed that chloride-cotransport blockade did not cause a significant desynchronization of action potential firing times in the CA3 subregion at the time that CA1 synchronous discharge was blocked but did reduce the number of action potentials associated with CA3 burst discharges. These data support our hypothesis that the anti-epileptic effects of chloride-cotransport antagonism in CA1 are mediated through the desynchronization of population activity. We hypothesize that interference with Na(+),K(+),2Cl(-) cotransport results in an increase in extracellular potassium ([K(+)](o)) that reduces the number of action potentials that are able to invade axonal arborizations and varicosities in all hippocampal subregions. This reduced efficacy of presynaptic action potential propagation ultimately leads to a reduction of synaptic drive and a desynchronization of the firing of CA1 pyramidal cells.

Embryogenesis of the central nervous system of the pond snail Lymnaea stagnalis L. An ultrastructural study.

The ultrastructural characteristics of the developing CNS of the pond snail, Lymnaea stagnalis, were investigated, with special attention paid to three specific stages of embryonic development, representing distinctly different phases of both the body morphogenesis and gangliogenesis. These were the 35% (veliger), the 50-55% (metamorphic), and the 75% (post-metamorphic, adult-like) stages of embryonic development. Also, a brief comparison was done with the CNS of hatchlings (100% of embryonic development). During embryogenesis specialized axo-axonic synapses and elements of the glial system, including the ganglionic (neural) sheath, were rarely observed, whereas the frequent occurrence of unspecialized axo-somatic contacts could be demonstrated. Synapse-like axo-axonic connections could be found first in 75% embryos, showing asymmetric vesicle clustering on the "presynaptic" side and increased electron density of the apposed membranes. These phenomena may reflect the dominance of modulatory processes in the CNS during embryogenesis, and the absence of the neural sheath may facilitate trophic and/or hormonal influences within the developing ganglia. The gradual increase in the size of ganglia and the diameter of their neuropils was not accompanied by any widening of the cell body layer or increasing diameter of the nerve cell bodies until the very end of embryogenesis. With respect to the ultrastructural organization of the neuropil, and possibly the entire CNS, a determining stage seems to be that of metamorphosis. Two types of neuropil could be observed at this time; the metamorphosing neuropil with an irregular organization of wavy axon profiles, and well-structured neuropil with a regular organization of axon profiles. Ganglia with irregular or regular neuropil occurred simultaneously in the developing CNS.

Connectivity of GABAergic calretinin-immunoreactive neurons in rat primary visual cortex.

In rat visual cortex neurons that are immunoreactive for the calcium-binding protein calretinin (CR+) constitute a distinct family which accounts for 17% of gamma-aminobutyric acid (GABA)-expressing cells. It is not clear, however, (i) whether CR is expressed exclusively in GABAergic neurons and (ii) how CR+ neurons are incorporated into neuronal circuits of rat visual cortex. To address these questions we studied synaptic relationships of CR+ neurons with GABA+ and GABA- elements in the neuropil of rat primary visual cortex (area 17). All CR+ neurons are nonpyramidal cells with smooth or sparsely spiny and often beaded dendrites. Of all CR+ neurons, 56% are located in layers 1 and 2/3. In layer 2/3, most CR+ neurons are bipolar-shaped and have vertically oriented dendrites. Many ascending dendritic branches reach layer 1 where they run parallel to pial surface. CR+ axons are thin, highly branched near the cell body and often send descending collaterals to layers 5 and 6. Double immunofluorescence labeling revealed GABA in 94% of CR+ cell bodies in layer 2/3. Electron microscopic analysis shows that all CR+ axon terminals contain elongated vesicles and form symmetric synapses. Postembedding staining shows that 98% of CR+ terminals are GABA+. GABA-immunoreactivity is also present in somata and thick dendrites of CR+ neurons but many thin dendrites are GABA-. CR+ somata, dendrites and axon terminals are enriched in mitochondria. Somata and thick CR+ dendrites are densely innervated. At least 68% of the targets of CR+ terminals in layer 2/3 are GABA+ and > or = 50% of these are other CR+ neurons. The remainder (32%) of targets of CR+ terminals are thin dendrites of GABA- cells. In contrast, in layers 5 and 6, 60% of CR+ terminals form synapses with GABA- somatic profiles. The preferential interactions of layer 2/3 CR+ neurons with GABAergic neurons, and with CR+ neurons in particular, suggests that these cells play a role in the inhibition of inhibitory neurons of the same layer. Through these interactions CR+ cells may reduce inhibition of pyramidal cells in layers 2/3, 5 and 6 and thus disinhibit a column of neurons.

GABAB-Receptor-mediated currents in interneurons of the dentate-hilus border.

GABA(B)-receptor-mediated inhibition was investigated in anatomically identified inhibitory interneurons located at the border between the dentate gyrus granule cell layer and hilus. Biocytin staining was used to visualize the morphology of recorded cells. A molecular layer stimulus evoked a pharmacologically isolated slow inhibitory postsynaptic current (IPSC), recorded with whole cell patch-clamp techniques, in 55 of 63 interneurons. Application of the GABA(B) receptor antagonists, CGP 35348 (400 microM) or CGP 55845 (1 microM) to a subset of 25 interneurons suppressed the slow IPSC by an amount ranging from 10 to 100%. In 56% of these cells, the slow IPSC was entirely GABA(B)-receptor-mediated. However, in the remaining interneurons, a component of the slow IPSC was resistant to GABA(B) antagonists. Subtraction of this antagonist resistant current from the slow IPSC isolated the GABA(B) component (IPSC(B)). This IPSC(B) had a similar onset and peak latency to that recorded from granule cells but a significantly shorter duration. The GABA(B) agonist, baclofen (10 microM), produced a CGP 55845-sensitive outward current in 19 of 27 interneurons. In the eight cells that lacked a baclofen current, strong or repetitive ML stimulation also failed to evoke an IPSC(B), indicating that these cells lacked functional GABA(B) receptor-activated potassium currents. In cells that expressed a baclofen current, the amplitude of this current was approximately 50% smaller in interneurons with axons that projected into the granule cell dendritic layer (22.2 +/- 5.3 pA; mean +/- SE) than in interneurons with axons that projected into or near the granule cell body layer (46.1 +/- 10.0 pA). Similarly, the IPSC(B) amplitude was smaller in interneurons projecting to dendritic (9.4 +/- 2.7 pA) than perisomatic regions (34.3 +/- 5.1 pA). These findings suggest that GABA(B) inhibition more strongly regulates interneurons with axons that project into perisomatic than dendritic regions. To determine the functional role of GABA(B) inhibition, we examined the effect of IPSP(B) on action potential firing and synaptic excitation of these interneurons. IPSP(B) and IPSP(A) both suppressed depolarization-induced neuronal firing. However, unlike IPSP(A), suppression of firing by IPSP(B) could be easily overcome with strong depolarization. IPSP(B) markedly suppressed N-methyl-D-aspartate but not AMPA EPSPs, suggesting that GABA(B) inhibition may play a role in regulating slow synaptic excitation of these interneurons. Heterogeneous expression of GABA(B) currents in hilar border interneurons therefore may provide a mechanism for the differential regulation of excitation of these cells and thereby exert an important role in shaping neuronal activity in the dentate gyrus.

The p38 mitogen-activated protein kinase inhibitor SB203580 antagonizes the inhibitory effects of interleukin-1β on long-term potentiation in the rat dentate gyrus in vitro

Levels of the pro-inflammatory cytokine interleukin-1β are known to be elevated in patients with chronic disorders such as Alzheimer's disease. We have investigated the effects of interleukin-1β on long-term potentiation and N-methyl- d-aspartate receptor-mediated field potentials in the rat dentate gyrus in vitro utilizing field extracellular recordings obtained from the middle third of the molecular layer of the dentate gyrus. Presynaptic stimulation was applied to the commissural/association pathway at a frequency of 0.05 Hz and at a distance of 50 μm from the granule cell body layer. As previously reported, interleukin-1β (1 ng/ml) caused an inhibition of long-term potentiation (108±2% of baseline 1 h following application of tetanic stimulation compared with 145±5% in vehicle control slices). This action of interleukin-1β on long-term potentiation, as well as an inhibition of N-methyl- d-aspartate receptor-mediated field potentials, was attenuated by pre-treatment of slices with the p38 mitogen-associated protein kinase inhibitor SB203580 (1 μM). SB203580 alone had no significant affect on long-term potentiation, but did cause an increase in baseline synaptic transmission [107±2% of baseline, 1 h after SB203580 (1 μM) treatment]. The p42/44 mitogen-activated protein kinase cascade inhibitor PD98059 (50 μM) did not inhibit the interleukin-1β-induced inhibition of N-methyl- d-aspartate receptor-mediated field potentials. The cyclooxygenase inhibitor indomethacin (50 μM) was found to attenuate the interleukin-1β-induced effects on both long-term potentiation and N-methyl- d-aspartate receptor-mediated field potentials. The lipid second messenger analogue C2 ceramide (20 μM) was found to attenuate the expression of long-term potentiation (108±3% of baseline 1 h following tetanic stimulation), and this effect was not blocked by pre-treatment with SB203580. To investigate a possible role for interleukin-1β in the normal expression of long-term potentiation, the interleukin-1 receptor antagonist (25 ng/ml) was applied during the maintenance phase of long-term potentiation. This was found to depress the sustained expression of long-term potentiation (116±6% of baseline 1 h following tetanic stimulation). Our results indicate possible signalling mechanisms by which interleukin-1β at pathophysiological concentrations may serve to inhibit long-term potentiation, and also suggests a role for IL-1β in the physiological expression of synaptic plasticity in the rat dentate gyrus in vitro.

Selective suppression of forward and recurrent "rapid" inhibition by local application of picrotoxin in area CA1 of rat hippocampal slices.

Living slices of rat hippocampus were used to study the possibility of suppressing forward and recurrent "rapid" inhibition in area CA1 by local application of picrotoxin, an antagonist of ionotropic gamma-aminobutyric acid (GABAA) receptors. Application of picrotoxin to the apical dendrites increased the duration of focal potentials recorded in the radial layer (143.0 +/- 7.5%, n = 5; here and subsequently, results are presented as mean +/- error of the mean and n is the number of experiments) but had no effect on the population peak in the pyramidal layer (103.0 +/- 19.6%, n = 5). This is evidence for the existence of suppression of direct, but not of recurrent inhibition. Application of picrotoxin to the cell body layer, on the other hand, significantly increased the population peak (654.5 +/- 245.1%, n = 4) and provoked convulsive activity in neurons, demonstrating suppression of recurrent inhibition. Local application of picrotoxin was further used to study the question of how completely antagonists of glutamate ionotropic receptors sensitive to alpha-amino-3-hydroxy-S-methyl-4-isoxazole propionic acid (AMPA) suppress inhibition in solutions with low magnesium contents. This question is important for interpreting experimental data obtained from measurements of the components of excitatory postsynaptic potentials (EPSP), which depend on activation of ionotropic glutamate receptors sensitive to N-methyl-d-aspartic acid (NMDA). A number of studies have suggested that even at low concentrations, AMPA receptor antagonists suppress forward inhibition to such an extent that it has no significant effect on measurements of the NMDA component of EPSP. Our data do not contradict this suggestion.

Opioid Receptor Subtype Expression Defines Morphologically Distinct Classes of Hippocampal Interneurons

The inhibition of hippocampal pyramidal cells occurs via inhibitory interneurons making GABAergic synapses on distinct segments of the postsynaptic membrane. In area CA1 of the hippocampus, the activation of mu- and delta-opioid receptors inhibits these interneurons, thereby increasing the excitability of the pyramidal cells. Through the use of selective opioid agonists and biocytin-filled whole-cell electrodes, interneurons possessing somata located within stratum oriens of hippocampal slices were classified according to the location of their primary axon termination and the expression of mu- or delta-opioid receptors. Activation of these opioid receptor subtypes resulted in outward currents in the majority of interneurons, which is consistent with their inhibition. Post hoc morphological analysis revealed that those interneurons heavily innervating the pyramidal cell body layer were much more likely to express mu-opioid receptors, whereas cells with axons ramifying in the pyramidal neuron dendritic layers were more likely to express delta-opioid receptors, as defined by the generation of outward currents. This morphological segregation of interneuron projections suggests that mu receptor activation would diminish GABA release onto pyramidal neuron somata, thereby increasing their excitability and output. Conversely, inhibition of interneurons via delta receptor activation would amplify afferent signaling to pyramidal neuron dendrites by reducing GABAergic inhibition of these structures.

Extracellular chloride and the maintenance of spontaneous epileptiform activity in rat hippocampal slices.

Previous studies showed that furosemide blocks spontaneous epileptiform activity without diminishing synaptic transmission or reducing hyperexcited field responses to electrical stimuli. We now test the hypothesis that the antiepileptic effects of furosemide are mediated through its blockade of the Na+,K+,2Cl- cotransporter and thus should be mimicked by a reduction of extracellular chloride ([Cl-]o). In the first set of experiments, field recordings from the CA1 cell body layer of hippocampal slices showed that spontaneous bursting developed within 10-20 min in slices perfused with low-[Cl-]o (7 mM) medium but that this spontaneous epileptiform activity ceased after a further 10-20 min. Intracellular recordings from CA1 pyramidal cells showed that normal action potential discharge could be elicited by membrane depolarization, even after the tissue was perfused with low-[Cl-]o medium for >2 h. In a second set of experiments, spontaneous bursting activity was induced in slices by perfusion with high-[K+]o (10 mM), bicuculline (100 microM), or 4-aminopyridine (100 microM). In each case, recordings from the CA1 region showed that reduction of [Cl-]o to 21 mM reversibly blocked the bursting within 1 h. Similar to previous observations with furosemide treatment, low-[Cl-]o medium blocked spontaneous hypersynchronous discharges without reducing synaptic hyperexcitability (i.e., hyperexcitable field responses evoked by electrical stimulation). In a third set of experiments, prolonged exposure (>1 h after spontaneous bursting ceased) of slices to systematically varied [Cl-]o and [K+]o resulted in one of three types of events: 1) spontaneous, long-lasting, and repetitive negative field potential shifts (7 mM [Cl-]o; 3 mM [K+]o); 2) oscillations consisting of 5- to 10-mV negative shifts in the field potential, with a period of approximately 1 cycle/40 s (16 mM [Cl-]o; 12 mM [K+]o); and 3) shorter, infrequently occurring negative field shifts lasting 20-40 s (21 mM [Cl-]o; 3 mM [K+]o). Our observations indicate that the effects of low [Cl-]o on neuronal synchronization and spontaneous discharge are time dependent. Similar effects were seen with furosemide and low [Cl-]o, consistent with the hypothesis that the antiepileptic effect of furosemide is mediated by the drug's effect on chloride transporters. Finally, the results of altering extracellular potassium along with chloride suggest that blockade of the Na+, K+,2Cl- cotransporter, which normally transports chloride from the extracellular space into glial cells, is key to these antiepileptic effects.

Embryonic development of theDrosophila brain. II. Pattern of glial cells

Glial cells in Drosophila and other insects are organized in an outer layer that envelops the surface of the central and peripheral nervous system (subperineurial glia, peripheral glia), a middle layer associated with neuronal somata in the cortex (cell body glia), and an inner layer surrounding the neuropile (longitudinal glia, midline glia, nerve root glia). In the ventral nerve cord, most glial cells are formed by a relatively small number of neuro-glioblasts; subsequently, glial cell precursors migrate and spread out widely to reach their final destination. By using a glia-specific marker (antibody against the Repo protein) we have reconstructed the pattern of glial cell precursors at successive developmental stages, focusing on the glia of the supraesophageal ganglion and subesophageal ganglion which are not described in previous studies. Digitized images of consecutive optical sections were used to generate 3-D models that show the spatial pattern of glial cell precursors in relationship to the neuropile, brain surface, and peripheral nerves. Similar to their spatial organization in the ventral nerve cord, glial cells of the brain populate the brain nerves and outer surface, cortical cell body layer, and cortex-neuropile interface. Neuropile-associated glial cells arise from a cluster located at the base of the supraesophageal ganglion; from this position, they migrate dorsally along the developing axon tracts and by late embryonic stages form a sheath around all neuropile compartments, including the supraesophageal commissure. Surface and cell body glial cells derive from several discrete foci, notably two large clusters at the deuterocerebrum/protocerebrum boundary and the posterior protocerebrum. From these foci, glial cells then fan out to envelop the surface of the supraesophageal ganglion.

Embryonic development of the Drosophila brain. II. Pattern of glial cells

Glial cells in Drosophila and other insects are organized in an outer layer that envelops the surface of the central and peripheral nervous system (subperineurial glia, peripheral glia), a middle layer associated with neuronal somata in the cortex (cell body glia), and an inner layer surrounding the neuropile (longitudinal glia, midline glia, nerve root glia). In the ventral nerve cord, most glial cells are formed by a relatively small number of neuro-glioblasts; subsequently, glial cell precursors migrate and spread out widely to reach their final destination. By using a glia-specific marker (antibody against the Repo protein) we have reconstructed the pattern of glial cell precursors at successive developmental stages, focusing on the glia of the supraesophageal ganglion and subesophageal ganglion which are not described in previous studies. Digitized images of consecutive optical sections were used to generate 3-D models that show the spatial pattern of glial cell precursors in relationship to the neuropile, brain surface, and peripheral nerves. Similar to their spatial organization in the ventral nerve cord, glial cells of the brain populate the brain nerves and outer surface, cortical cell body layer, and cortex-neuropile interface. Neuropile-associated glial cells arise from a cluster located at the base of the supraesophageal ganglion; from this position, they migrate dorsally along the developing axon tracts and by late embryonic stages form a sheath around all neuropile compartments, including the supraesophageal commissure. Surface and cell body glial cells derive from several discrete foci, notably two large clusters at the deuterocerebrum/protocerebrum boundary and the posterior protocerebrum. From these foci, glial cells then fan out to envelop the surface of the supraesophageal ganglion. J. Comp. Neurol. 402:32–47, 1998. © 1998 Wiley-Liss, Inc.