The effect of type 1 astrocytes on neuronal complexity: a fractal analysis.

Embryonic, ventral spinal cord neurons were grown on poly(d-lysine) (PDL) or on a monolayer of type 1 astrocytes. At various times from 6 h to 2 weeks postplating, cells were fluorescently labeled and fixed with 4% paraformaldehyde. The cell surface immunoreaction allowed visualization of neurons in their entirety, namely, cell bodies and various membranous extensions that included lamellipodia, growth cones, axons, and dendrites. Outlines were drawn for individual neurons and their fractal dimension (D) was calculated. Neurons on poly(d-lysine) reached a peak D at 3 days in vitro, 1 day later than neurons on astrocytes (2 days in vitro). The maximum D was greater for cells on poly(d-lysine) when compared with neurons on astrocytes. In a second experiment the maximum D was similar for neurons on both surfaces but neurons on PDL maintained a higher D for a much longer period than neurons on astrocytes. An examination of fluorescent images revealed that neurons on poly(d-lysine) exhibited lamellipodia and large growth cones for several days and these structures were likely responsible for the high D seen in these cells. These structures were rarely observed in neurons plated on astrocytes. Interestingly, D on both surfaces decreased to a similar value at between 1 and 2 weeks in vitro. The trend for D in these cultures, an initial increase to a peak value followed by a decrease to a stable value, is discussed in light of the chemical nature of the two surfaces and synapse formation and stabilization.

ets-2 is a target for an akt (Protein kinase B)/jun N-terminal kinase signaling pathway in macrophages of motheaten-viable mutant mice.

The transcription factor ets-2 was phosphorylated at residue threonine 72 in a colony-stimulating factor 1 (CSF-1)- and mitogen-activated protein kinase-independent manner in macrophages isolated from motheaten-viable (me-v) mice. The CSF-1 and ets-2 target genes coding for Bcl-x, urokinase plasminogen activator, and scavenger receptor were also expressed at high levels independent of CSF-1 addition to me-v cells. Akt (protein kinase B) was constitutively active in me-v macrophages, and an Akt immunoprecipitate catalyzed phosphorylation of ets-2 at threonine 72. The p54 isoform of c-jun N-terminal kinase-stress-activated kinase (JNK- SAPK) coimmunoprecipitated with Akt from me-v macrophages, and treatment of me-v cells with the specific phosphatidylinositol 3-kinase inhibitor LY294002 decreased cell survival, Akt and JNK kinase activities, ets-2 phosphorylation, and Bcl-x mRNA expression. Therefore, ets-2 is a target for phosphatidylinositol 3-kinase-Akt-JNK action, and the JNK p54 isoform is an ets-2 kinase in macrophages. Constitutive ets-2 activity may contribute to the pathology of me-v mice by increasing expression of genes like the Bcl-x gene that promote macrophage survival.

Persistent activation of GABA(A) receptor/Cl(-) channels by astrocyte-derived GABA in cultured embryonic rat hippocampal neurons.

Whole cell patch-clamp recordings using Cl(-)-filled pipettes revealed more negative levels of baseline current and associated current variance in embryonic rat hippocampal neurons co-cultured on a monolayer of astrocytes than those cultured on poly-D-lysine. These effects were mimicked by culturing neurons on poly-D-lysine in astrocyte-conditioned medium (ACM). The baseline current and variance decreased immediately in all cells after either local perfusion with saline or exposure to bicuculline, an antagonist of GABA at GABA(A) receptor/Cl(-) channels. Baseline current and variance in all cells reached a nadir at approximately 0 mV, the calculated equilibrium potential for Cl(-). Perfusion of ACM rapidly induced a sustained current in neurons, which also reversed polarity at approximately 0 mV. Bicuculline attenuated or eliminated the ACM-induced current at a concentration that completely blocked micromolar GABA-induced current. Quantitative analyses of spontaneously occurring fluctuations superimposed on the ACM-induced current revealed estimated unitary properties of the underlying channel activity similar to those calculated for GABA's activation of GABA(A) receptor/Cl(-) channels. Bicuculline-sensitive synaptic-like transients, which reversed at approximately 0 mV, were also detected in neurons cultured in ACM, and these were immediately eliminated along with the negative baseline current and superimposed current fluctuations by perfusion. Furthermore bicuculline-sensitive synaptic-like transients were rapidly and reversibly triggered when ACM was acutely applied. ACM induced an increase in cytoplasmic Ca(2+) in cultured embryonic hippocampal neurons that was completely blocked by bicuculline and strychnine. We conclude that astrocytes release diffusible substances, most likely GABA, that persistently activate GABA(A) receptor/Cl(-) channels in co-cultured neurons.

GABA receptor antagonists modulate postmitotic cell migration in slice cultures of embryonic rat cortex.

Recent studies indicate that GABA acts as a chemoattractant during rat cortical histogenesis. In vivo, GABA localizes in appropriate locations for a chemoattractant, along migratory routes and near target destinations for migrating cortical neurons. In vitro, GABA induces dissociated embryonic cortical neurons to migrate. Here, embryonic rat cortical slices were cultured in the presence or absence of GABA receptor (GABA-R) antagonists to assess GABA's effects on neuronal migration in situ. Gestational day 18 (E18) cortical slices were incubated overnight in bromodeoxyuridine (BrdU)-containing medium to label ventricular zone (vz) cells as they underwent terminal mitosis. The slices were then cultured in BrdU-free medium with or without GABA-R antagonists. In control slices, most BrdU(+) cells were observed in the cortical plate (cp) after 48 h. In contrast, cultures maintained in either saclofen (a GABA(B)-R antagonist) or picrotoxin (a GABA(A/C)-R antagonist) had few BrdU-labeled cp cells. However, the effects of the two antagonists were distinct. In the picrotoxin-treated slices, nearly half of all BrdU(+) cells remained in the vz and subventricular zone (svz), whereas saclofen treatment resulted in an accumulation of BrdU(+) cells in the intermediate zone (iz). Bicuculline, a GABA(A)-R antagonist, did not block, but rather enhanced migration of BrdU(+) cells into the cp. These results provide evidence that picrotoxin-sensitive receptors promote the migration of vz/svz cells into the iz, while saclofen-sensitive receptors signal cells to migrate into the cp. Thus, as cortical cells differentiate, changing receptor expression appears to modulate migratory responses to GABA.

Surface-accessible GABA supports tonic and quantal synaptic transmission.

Exocytosis is commonly viewed as the only secretory process able to account for quantal forms of fast synaptic transmission. However, the demonstrated variability and composite properties of miniature postsynaptic signals are not easily explained by all-or-none exocytotic discharge of transmitter in solution from inside vesicles. Recent studies of endocrine secretion have shown that hormone release does not coincide with exocytosis due to its trapping in the core matrix of the granule. Thus, we tested whether the synaptic transmitter GABA could also be held in a matrix before being released. Using confocal microscopy and flow cytometry of embryonic rat hippocampal neurons, we found a GABA immunoreaction at the surface of live cell bodies and growth cones that coincided spatially and quantitatively with the binding of tetanus toxin fragment C (TTFC). TTFC binds predominantly at membrane sites containing the trisialoglycosphingolipid GT1b. Using flow cytometry, GT1b-containing liposomes preincubated in 100 nM GABA exhibited the same relationship between GABA and TTFC surface binding as found on neurons and growth cones. Embryonic neurons differentiated in culture expressed initially a tonic, and after 3-5 days, transient, postsynaptic signals mediated by GABA acting at GABA(A) receptor/Cl(-) channels. A stream of saline applied to the neuronal surface rapidly and reversibly suppressed both tonic and transient signals. A brief application of the GABAmimetic isoguvacine immediately transformed both tonic and transient GABAergic signals into tonic and transient isoguvacinergic signals. These results and those in the literature are consistent with an immediately releasable compartment of transmitter accessible from the presynaptic surface.

Synaptic connectivity in hippocampal neuronal networks cultured on micropatterned surfaces.

Embryonic rat hippocampal neurons were grown on patterned silane surface in order to organize synapse formations in a controlled manner. The surface patterns were composed of trimethoxysilylpropyl-diethylenetriamine (DETA) lines separated by tridecafluoro-1,1,2,2-tetrahydrooctyl-1-dimethylchlorosilane (13F) spaces. Pre- and post-synaptic specializations were identified by immunostaining for synapsin I and microtubule-associated protein-2 (MAP-2). Functional synaptic connections were examined by recording simultaneously from pairs of neurons using the whole-cell configuration of the patch-clamp technique. Spontaneous and evoked synaptic currents were recorded in neurons cultured for 2-14 days. The formation of functional connections was accompanied by the appearance of spontaneous synaptic currents (SSCs), which could be detected after approximately 3 days in culture in the absence of evoked synaptic currents (ESCs). ESCs were detected only after approximately 7 days in culture, mostly in the form of unidirectional synaptic connections. Other forms of synaptic connectivity, such as bidirectional and autaptic connections, were also identified. Both transient GABAergic and glutamatergic signals mediated the transmissions between communicating cells. These results demonstrate the combination of various types of synaptic connections forming simple and complex networks in neurons cultured on line (DETA)-space (13F) patterns. Finally, precisely synchronized SSCs were recorded in neuron pairs cultured on pattern indicating the existence of a fast-acting feedback mechanism mediated by pre-synaptic GABA(A) receptors.

Astrocytes regulate the developmental appearance of GABAergic and glutamatergic postsynaptic currents in cultured embryonic rat spinal neurons.

The effects of astrocytes on the emergence of synaptic transients and excitable membrane properties in cultured, embryonic, rat ventral spinal neurons were studied with electrical and optical recording techniques. Neurons on astrocytes had significantly longer neurites and an accelerated rate of growth in surface membrane during the initial 24 h in culture compared to neurons on poly-D-lysine (PDL). GABAergic (GABA, gamma-aminobutyric acid) and glutamatergic transients appeared spontaneously in co-cultured neurons by 24 h. GABAergic quanta did not appear in neurons on PDL until 4 days in culture, and glutamatergic transients did not emerge until 7 days in culture. Astrocyte-conditioned medium (ACM) partially mimicked the effects of direct astrocytic contact. GABAergic transients appeared by 2 days, and glutamatergic signals by 4 days in neurons on PDL exposed to ACM. All of the spontaneous, synaptic-like transients were eliminated by tetrodotoxin or Ca2+o-free saline, implicating voltage-dependent cation channels in their generation. Astrocytes immediately and significantly increased the density of voltage-dependent Na+ currents compared to neurons on PDL, but by the end of 24 h, Na+ current densities were identical. Electrophysiological and optical recording revealed comparable densities of high-voltage-activated (HVA) Ca2+ currents on both co-cultured neurons and neurons on PDL throughout the first week. However, neurons on astrocytes had significantly greater contributions of P/Q-type currents and lesser contributions of L-type currents beginning at 24 h and continuing for 7 days. The contribution of N-type current was significantly more in co-cultured neurons only at 24 h. Thus, in vitro, astrocytes help to differentiate specific excitable membrane properties in spinal neurons, along with GABAergic and glutamatergic forms of synaptic transmission.

Distribution of GABA(C)-like responses among acutely dissociated rat retinal neurons.

GABAergic responses of acutely dissociated rat retinal neurons, including both bipolar cells (BCs) and other, morphologically round cells (RCs), were assayed with the fluorescent (FL), voltage-sensitive probe oxonol DiBaC4(5). Using intensified video microscopy and simultaneous recording, GABA responses were identified in one-third of cells in a typical microscope field; of these 85% hyperpolarized (0.05-0.3 log unit FL decreases) while the remainder depolarized (0.05-0.2 log unit FL increases). GABA-sensitive cells were also TACA-sensitive (trans-4-Aminocrotonic acid), and these ligands appeared interchangeable in ability to evoke responses. In RCs, an asymmetric co-responsive pattern was observed between GABA- and muscimol-evoked events. Muscimol-sensitive RCs responded well to GABA, but not all GABA-sensitive RCs responded to muscimol. In GABA-sensitive BCs, muscimol responses were typically weak or absent. Few BCs or RCs responded to CACA (cis-4-Aminocrotonic acid). Bicuculline-resistant GABA responses occurred in approximately 80% of GABA-responsive RCs and BCs. Both bicuculline-sensitive (GABA(A)-like) and bicuculline-insensitive (GABAc-like) responses were resistant to picrotoxin. Although a small minority of GABA-sensitive cells hyperpolarized in response to R(+)baclofen, bicuculline-insensitive responses were not antagonized by 2-hydroxysaclofen, and were abolished in low [Cl-]o. Results suggested (1) that bicuculline-insensitive, Cl(-)-dependent, GABAc-like responses were broadly distributed and predominant among dissociated rat retinal neurons; (2) that muscimol was a particularly weak agonist for rat retinal BCs; and (3) that oxonol was a sensitive probe for retinal GABA responses.

Central neuronal synapse formation on micropatterned surfaces.

Controlling synapse formation is a key to patterning of neurons into functional circuits and networks in vitro. However, the process of synapse formation among neurons grown on artificial surfaces is relatively unstudied. We cultured embryonic hippocampal cells on trimethoxysilylpropyl-diethylenetriamine (DETA) and tridecafluoro-1, 1,2,2-tetrahydrooctyl-1-dimethylchlorosilane (13F), and on patterns composed of DETA lines separated by 13F spaces. For comparison, neurons were concurrently plated on surfaces coated with uniform poly-d-lysine (PDL). Pre- and postsynaptic specializations were identified by immunostaining for synapsin I and microtubule-associated protein-2 (MAP-2). Spontaneous (SPCs) and evoked (EPCs) postsynaptic currents were recorded using dual patch-clamp techniques. We found that DETA promoted synapse formation, whereas evidence for synapse formation on 13F was barely detected. MAP-2+ neuronal soma and rapidly growing dendrites were co-localized with synapsin I puncta faithfully along DETA lines. The expression of synapsin I puncta, and MAP-2+ soma and dendrites correlated well with the appearance of SPCs. Synapsin I, MAP-2 and SPCs emerged together at days 3-4 and increased at day 7, when EPCs appeared. Synaptic signals occurring during 4-7 days in culture were all GABAergic. These results indicate that fully functional synapses are formed on silane surfaces, demonstrating the suitability of patterned silane surfaces for organizing synapse formation in vitro.

GABAergic cells and signals in CNS development.

GABA is formed primarily from decarboxylation of glutamate by a family of cytosolic and membrane-bound GAD enzymes. In the adult, GAD-derived GABA sustains the vitality of the central nervous system (CNS), since blockage of GAD rapidly leads to convulsions and death. In plants, cytosolic GAD synthesizes GABA in response to hormones and environmental stress. Since decarboxylation involves protonation, secretion of GABA serves to buffer cytosolic pH in plant cells. Families of GAD and GABAA receptor/Cl- channel transcripts and encoded proteins emerge early and seemingly everywhere during CNS development, with their abundance closely paralleling neurogenesis and peaking before birth. Micromolar GABA acts at receptor/Cl-channels to depolarize progenitor cells in the cortical neuroepithelium; it also elevates their cytosolic Ca2+ (Cac2+) levels. In some way, these effects decrease proliferation. GABA directs the migration of postmitotic neuroblasts at femtomolar concentrations and stimulates their random motility at micromolar concentrations via Ca2+ signaling mechanisms. Activation of GABAA receptors by micromolar GABA may limit motility via membrane depolarization and elevated Cac2+. These results indicate that in vitro GABA can affect embryogenesis of the CNS through effects on cell proliferation and migration. As neurons differentiate postnatally, Cl(-)-dependent depolarization disappears together with GABAergic Cac2+ signals. Physiologically occurring GABAergic signals at Cl-channels exist in tonic and transient forms. Since the former are found on progenitor cells while both are present in postmitotic neurons, mechanisms to generate transients differentiate in the latter. Surprisingly, tonic and transient forms of GABAergic signaling at Cl-channels are rapidly and smoothly interconvertible and seem to be derived from online GABA synthesis in a surface-accessible compartment of the membrane.

Differential response of cortical plate and ventricular zone cells to GABA as a migration stimulus.

A microdissection technique was used to separate differentiated cortical plate (cp) cells from immature ventricular zone cells (vz) in the rat embryonic cortex. The cp population contained >85% neurons (TUJ1(+)), whereas the vz population contained approximately 60% precursors (nestin+ only). The chemotropic response of each population was analyzed in vitro, using an established microchemotaxis assay. Micromolar GABA (1-5 microM) stimulated the motility of cp neurons expressing glutamic acid decarboxylase (GAD), the rate-limiting enzyme in GABA synthesis. In contrast, femtomolar GABA (500 fM) directed a subset of GAD- vz neurons to migrate. Thus, the two GABA concentrations evoked the motility of phenotypically distinct populations derived from different anatomical regions. Pertussis toxin (PTX) blocked GABA-induced migration, indicating that chemotropic signals involve G-protein activation. Depolarization by micromolar muscimol, elevated [K+]o, or micromolar glutamate arrested migration to GABA or GABA mimetics, indicating that migration is inhibited in the presence of excitatory stimuli. These results suggest that GABA, a single ligand, can promote motility via G-protein activation and arrest attractant-induced migration via GABAA receptor-mediated depolarization.

Embryonic rat hippocampal neurons and GABAA receptor subunit-transfected non-neuronal cells release GABA tonically.

We used patch-clamp recording techniques to investigate the contribution of GABA to baseline membrane properties in cultured embryonic rat hippocampal neurons. Almost all of the neurons recorded with Cl--filled pipettes and clamped at negative potentials exhibited baselines that were noticeably noisy, with microscopic fluctuations superimposed on the macroscopic holding current. A gentle steam of saline applied to the neuronal surface rapidly and reversibly reduced the baseline current and fluctuations, both of which were completely eliminated by bicuculline. Fluctuation analysis showed that the variance in the baseline current signal was exponentially distributed with estimated kinetics comparable to those activated by submicromolar concentrations of exogenous GABA. The kinetics of Cl- channels activated by endogenous GABA displayed a potential sensitivity comparable to those activated by exogenous GABA. Non-neuronal cells stably transfected with alpha1 and gamma2 GABAA receptor subunits exhibited little baseline current variance when recorded with Cl--filled pipettes. Addition of micromolar GABA to the extracellular saline or to the pipette solution induced a saline- and bicuculline-sensitive baseline current signal comparable to that recorded in hippocampal neurons. Thus, both intra- and extracellular sources of GABA could contribute to the baseline properties recorded in these cultured neurons.

Microlithographic determination of axonal/dendritic polarity in cultured hippocampal neurons.

High resolution substrates, created using patterned self-assembled monolayers, are shown to direct axonal and dendritic process extension at the level of a single hippocampal neuron. Axons and dendrites were identified using morphological characteristics and immunocytochemical markers. Patterns were formed on glass coverslips from a co-planar monolayer of cell adhesive aminosilanes and non-adhesive fluorinated silanes. On patterned surfaces, the percentage of the total number of cells attached to the 0.71 mm2 substrate field with compliance to the 25-micron diameter 'somal adhesion site' reached 41 +/- 7% (mean +/- S.D., 428 cells counted). A total of 76 +/- 11% of cells that adhered to a somal attachment site developed a lone process > or = 100 microns oriented in the direction of the continuous aminosilane pathway which was shown to express axonal markers. Cells on either the fluorinated silane, which is non-permissive for neurite outgrowth, or localized on an aminosilane region only 5 microns wide failed to extend major processes. This approach is amenable to a variety of industry standard fabrication techniques and may be used to study the role of fine scale spatial cues in neuronal development and synapse formation.

Kainate induces an intracellular Na+-activated current in cultured embryonic rat hippocampal neurones.

1. In embryonic rat hippocampal neurones cultured for < 3 days, kainate induced an inward current at negative potentials that recovered to baseline levels immediately upon termination of agonist application. However, in neurones cultured for longer, the kainate-induced current was often followed by a long-lasting inward current that slowly recovered to baseline levels. The amplitude of the delayed current (Idelay) triggered by kainate was positively related both to the duration of application at constant agonist concentration and to concentration at constant application duration. 2. Idelay could last for several minutes and was accompanied by a conductance increase, which closely paralleled current amplitude. Depression of the kainate-induced current response at receptor level with CNQX or at ionic level with Na+-free solution eliminated Idelay. However, when applied during Idelay neither CNQX nor Na+-free solution had any effect on Idelay. Li+ effected the same response as Na+ in mediating kainate-induced Idelay. 3. GABA-activated Cl- current, which was associated with the same amount of inwardly directed charge flow at the same potential as that induced by kainate, did not trigger a long-lasting delayed current. 4. Idelay depended on the existence of extracellular K+ and its amplitude increased with the increase in K+ concentration. Neither applying Cl-- or Ca2+-free solutions nor increasing intracellular Ca2+ buffering speed and capacity altered Idelay. Exposure to the specific KCa channel blockers apamin and charybdotoxin also failed to alter Idelay. However, Idelay could be blocked by Cs+, Ba2+ and high concentrations of 4-aminopyridine (4-AP) and TEA. 5. Inside-out excised patch-clamp recordings revealed that low density or highly clustered Na+-activated K+ channels were expressed in the cell bodies of cultured embryonic rat hippocampal neurones. These could be the elementary channels underlying Idelay.

Astrocytes regulate developmental changes in the chloride ion gradient of embryonic rat ventral spinal cord neurons in culture.

1. Embryonic rat ventral spinal cord neurons were dissociated at day 15 and grown on: (i) poly-D-lysine (PDL); (ii) a confluent monolayer of type I astrocytes; or (iii) PDL in astrocyte-conditioned medium (ACM) to examine the influence of astroglia on the regulation of GABAA receptor/Cl- channel properties. 2. Potentiometric oxonol dye recordings of intact cells indicated that embryonic neurons were uniformly depolarized by muscimol. The depolarizing effects disappeared in cells dissociated during the early postnatal period and recovered in culture for 24 h. Similar recordings using the calcium-imaging dye fura-2 AM revealed that GABA or muscimol triggered a sustained rise in cytosolic Ca2+ (Ca2+c ) in embryonic neurons that was dependent on extracellular Ca2+, blocked by bicuculline and nifedipine and sensitive to changes in extracellular chloride. The incidence and amplitude of the Ca2+ response decreased with time in vitro and was accelerated in neurons cultured on astrocytes compared with those on PDL. 3. Perforated patch-clamp recordings revealed that GABA depolarized neurons in a Cl--dependent and bicuculline-sensitive manner. Both the resting membrane potential and the GABA equilibrium potential became more hyperpolarized with time in vitro. 4. Astrocytes and ACM accelerated the transformation of GABAergic potential responses from depolarizing to hyperpolarizing. The change occurred over the first 4 days in co-culture or in ACM but took more than 2 weeks in neurons cultured on PDL alone. 5. The intrinsic, elementary properties of GABAA receptor/Cl- channels including open time and unitary conductance changed independently of the presence of astrocytes or ACM. Mean open time of the dominant kinetic component decreased and conductance increased with time in vitro. 6. In sum, astrocytes accelerate the developmental change in the Cl- ion gradient extrinsic to GABAA receptor/Cl- channels, which is critical for triggering Ca2+ entry, without influencing parallel changes in the intrinsic properties of the channels.

Astrocyte-conditioned saline supports embryonic rat hippocampal neuron differentiation in short-term cultures.

Embryonic rat hippocampal neurons were cultured for 1-2 days in serum-free, HEPES-buffered Tyrode's solution. The effects of cortical astrocytes and astrocyte-conditioned saline on neuron survival, membrane surface area and the expression of functional amino acid neurotransmitter receptors were studied. Neurons grown in Tyrode's solution alone survived well for 1 day but deteriorated thereafter both in terms of percent neurons surviving and the amplitudes and densities of GABA-, glycine-, kainate-and NMDA-induced currents. Neurons grown in Tyrode's previously conditioned by astrocytes for 24 h had significantly larger apparent plasma membrane surface area, as indexed by whole-cell membrane capacitance, and larger amplitudes and densities of the amino acid-induced currents after both 1 and 2 days. The survival rate and neurite outgrowth were also greater in the astrocyte-conditioned saline group after 2 days in culture. Similarly, neurons cultured on glass cover-slips facing a confluent monolayer of astrocyte were larger in apparent plasma membrane area and amino acid-induced currents than neurons cultured in Tyrode's alone. Neurons cultured in saline conditioned by astrocytes provide a strategy to study the physiological basis of astrocyte-directed neuronal differentiation in the absence of ambiguities arising from the inclusion of sera and other additives often used in vitro.

Astrocytes regulate amino acid receptor current densities in embryonic rat hippocampal neurons.

Embryonic rat hippocampal neurons were cultured in a serum-free defined medium (MEM/N3) either directly on poly-D-lysine (PDL) or on a confluent monolayer of postnatal cortical astrocytes, C6 glioma cells, or Rat2 fibroblasts. Neurons on PDL were grown in MEM/N3 or in MEM/N3 conditioned for 24 h by astrocytes or C6 cells. Membrane capacitance (Cm) and gamma-aminobutyric acid (GABA)-, glycine-, kainate-, and N-methyl-D-aspartate (NMDA)-induced currents were quantified using whole-cell patch-clamp recordings. Cm as well as the amplitude and the density of these currents in neurons cultured on astrocytes were significantly greater than those in neurons grown on PDL after 24 and 48 h. C6 cells mimicked astrocytes in promoting Cm and GABA-, glycine-, and NMDA-evoked, but not kainate-evoked, currents. Cm and currents in neurons grown on Rat2 cells were comparable to those in neurons on PDL. Astrocytes maintained in culture for 3 months were noticeably less effective than freshly prepared ones just grown to confluence. Suppression of spontaneous cytoplasmic Ca2+ (Ca[c]2+) elevations in astrocytes by 1,2-bis(2-aminophenoxy) ehane-N, N, N, N-tetraacetic acid acetoxymethyl ester (BAPTA-AM) loaded intracellularly blocked the observed modulatory effects. Medium conditioned by either astrocytes or C6 cells mimicked the effects of direct coculture of neurons on these cells in promoting Cm and amino acid-evoked currents. Inclusion of antagonists at GABA and glutamate receptors in coculture experiments blocked the observed effects. Thus, diffusible substances synthesized and/ or secreted by astrocytes in a Ca(c)2+-dependent manner can regulate neuronal growth and aminoacid receptor function, and these effects may involve neuronal GABA and glutamate receptors.

Proton-induced cation current in embryonic rat spinal cord neurons changes ion dependency over time in vitro.

A rapid increase in proton concentration [H+]0 induces Na+ conductance in a variety of cell types. Here we report that H+ trigger a cation-selective channel whose ion dependency changes over time in culture. Whole-cell recordings of ventral spinal cord neurons dissociated at E15 and cultured for up to 14 days revealed that more than 80% had H(+)-induced inward current responses exhibiting a rapid decay phase. The current response was activated beginning about pH 6.8. Following decay, several minutes were required for complete recovery. More modest decreases in pH, which by themselves failed to activate this current, depressed those triggered by effective changes in pH. The currents recorded from cells in culture for less than 7 days could be abolished completely in the absence of Ca2+ and persisted in Na(+)-free and Ba(2+)-containing solutions. Ensemble analysis of current fluctuations recorded at the peak of the current allowed us to estimate a unitary channel conductance of 7.0 pS and a mean open time of 4.1 ms. In neurons cultured 2 weeks or more, protons induced an inward current response with similar kinetic properties, but with [Na+]0 dependency. Thus, proton-activated cation conductance in embryonic rat spinal cord neurons is self-limiting and involves brief openings of cation-selective channels whose ion dependency changes over time in culture.

GABA induces GABAergic MSCs in cultured embryonic rat thalamic neurons.

Application of 0.1-10 microM GABA in the vicinity of cultured embryonic rat thalamic neurons recorded with patch pipettes in the presence of 2 microM TTX induced or increased the frequency of miniature synaptic currents (MSCs) that reversed polarity at the Cl- equilibrium potential. These MSCs were blocked by the GABAA receptor antagonist bicuculline and exhibited exponential decay kinetics that closely paralleled those estimated from fluctuation analysis of Cl- channels activated pharmacologically by applying 1-10 microM GABA to the same cells. We conclude that the MSCs are mediated by GABA. Application of the GABAA receptor agonist muscimol activated Cl- current but failed to induce GABAergic MSCs while submicromolar concentrations of GABA evoked GABAergic MSCs but did not activate Cl- channels. The GABAB receptor agonist (-)baclofen did not mimic GABA in inducing MSCs. Induction of GABAergic MSCs by GABA required extracellular Ca2+. Verapamil and Co2+, which block voltage-dependent calcium channels, completely blocked GABA-induced MSCs independent of their effects on the direct activation of a Cl- current response. The results indicate that GABA can trigger GABAergic Cl(-)-dependent MSCs in a Cao(2+)-dependent manner. The mechanism may involve a novel receptor and/or signal transduction pathway.

Early upregulation of medium neurofilament gene expression in developing spinal cord of the wobbler mouse mutant.

Homozygous wobbler mouse mutants develop a progressive paralysis due to spinal motoneuron degeneration. To understand the molecular aspect underlying the genetic defect we have studied the embryonic (from E13) and postnatal expression of the three neurofilament and choline acetyltransferase genes in each member from several wild-type (wt) and wobbler (wr) progenies. There are no variations among wt littermates at all ages studied. In contrast, analyses of neurofilament mRNA reveals a 3-4-fold increase of medium neurofilament (NFM) mRNA in wobbler mice (wr/wr). The pattern of increased NFM mRNA during development, prior to the appearance of the wobbler phenotype, among littermates (from heterozygous carriers) conforms to a mendelian inheritance of a single gene defect 1:2:1 (wr/wr:wr/+:+/+). Light and heavy neurofilament mRNA levels are also increased later in development exclusively in those individuals with high NFM mRNA values indicating that increase of the latter is associated with increase of the light and heavy subunit expression. Also NF proteins are increased. Expression of choline acetyltransferase gene is instead always comparable to normal control. Our study provides novel insights into the nature of the wobbler defect, strengthening the hypothesis that neurofilament accumulation plays a pivotal role in the etiopathogenesis of motoneuron degeneration.