Altered balance of glutamatergic/GABAergic synaptic input and associated changes in dendrite morphology after BDNF expression in BDNF-deficient hippocampal neurons.

Cultured neurons from bdnf-/- mice display reduced densities of synaptic terminals, although in vivo these deficits are small or absent. Here we aimed at clarifying the local responses to postsynaptic brain-derived neurotrophic factor (BDNF). To this end, solitary enhanced green fluorescent protein (EGFP)-labeled hippocampal neurons from bdnf-/- mice were compared with bdnf-/- neurons after transfection with BDNF, bdnf-/- neurons after transient exposure to exogenous BDNF, and bdnf+/+ neurons in wild-type cultures. Synapse development was evaluated on the basis of presynaptic immunofluorescence and whole-cell patch-clamp recording of miniature postsynaptic currents. It was found that neurons expressing BDNF::EGFP for at least 16 h attracted a larger number of synaptic terminals than BDNF-deficient control neurons. Transfected BDNF formed clusters in the vicinity of glutamatergic terminals and produced a stronger upregulation of synaptic terminal numbers than high levels of ambient BDNF. Glutamatergic and GABAergic synapses reacted differently to postsynaptic BDNF: glutamatergic input increased, whereas GABAergic input decreased. BDNF::EGFP-expressing neurons also differed from BDNF-deficient neurons in their dendrite morphology: they exhibited weaker dendrite elongation and stronger dendrite initiation. The upregulation of glutamatergic synaptic input and the BDNF-induced downregulation of GABAergic synaptic terminal numbers by postsynaptic BDNF depended on tyrosine receptor kinase B activity, as deduced from the blocking effects of K252a. The suppression of dendrite elongation was also prevented by block of tyrosine receptor kinase B but required, in addition, glutamate receptor activity. Dendritic length decreased with the number of glutamatergic contacts. These results illuminate the role of BDNF as a retrograde synaptic regulator of synapse development and the dependence of dendrite elongation on glutamatergic input.

Brain-derived neurotrophic factor modulates GABAergic synaptic transmission by enhancing presynaptic glutamic acid decarboxylase 65 levels, promoting asynchronous release and reducing the number of activated postsynaptic receptors.

Brain-derived neurotrophic factor is known to modulate the function of GABAergic synapses, but the site of brain-derived neurotrophic factor action is still a matter of controversy. This study was aimed at further dissecting the functional alterations produced by brain-derived neurotrophic factor treatment of GABAergic synaptic connections in cultures of the murine superior colliculus. The functional consequences of long-term brain-derived neurotrophic factor treatment were assessed by analysis of unitary evoked and delayed inhibitory postsynaptic currents in response to high frequency stimulation of single axons. It was found that brain-derived neurotrophic factor facilitated the asynchronous release, but had no effect on the probability of evoked release, the size of the readily releasable pool, and the paired-pulse behavior of evoked inhibitory postsynaptic currents. However, the amplitudes of evoked inhibitory postsynaptic currents, delayed inhibitory postsynaptic currents and miniature inhibitory postsynaptic currents were significantly reduced. Non-stationary fluctuation analysis revealed a decrease in the open channel number at the miniature/evoked inhibitory postsynaptic current peak, but no effect on the mean GABA(A) receptor single channel conductance. Quantitative immunocytochemistry uncovered a significant elevation of presynaptic levels of glutamic acid decarboxylase 65. Together, these findings indicate that brain-derived neurotrophic factor treatment induces pre- as well as postsynaptic changes. What effect predominates will depend on the presynaptic activity pattern: at low activation rates brain-derived neurotrophic factor-treated synapses display a pronounced postsynaptic depression, but at high frequencies this depression is fully compensated by an enhancement of asynchronous release.

Pathological gamma oscillations, impaired dopamine release, synapse loss and reduced dynamic range of unitary glutamatergic synaptic transmission in the striatum of hypokinetic Q175 Huntington mice.

Huntington's disease (HD) is a severe genetically inherited neurodegenerative disorder. Patients present with three principal phenotypes of motor symptoms: choreatic, hypokinetic-rigid and mixed. The Q175 mouse model of disease offers an opportunity to investigate the cellular basis of the hypokinetic-rigid form of HD. At the age of 1 year homozygote Q175 mice exhibited the following signs of hypokinesia: Reduced frequency of spontaneous movements on a precision balance at daytime (-55%), increased total time spent without movement in an open field (+42%), failures in the execution of unconditioned avoidance reactions (+32%), reduced ability for conditioned avoidance (-96%) and increased reaction times (+65%) in a shuttle box. Local field potential recordings revealed low-frequency gamma oscillations in the striatum as a characteristic feature of HD mice at rest. There was no significant loss of DARPP-32 immunolabeled striatal projection neurons (SPNs) although the level of DARPP-32 immunoreactivity was lower in HD. As a potential cause of hypokinesia, HD mice revealed a strong reduction in striatal KCl-induced dopamine release, accompanied by a decrease in the number of tyrosine hydroxylase-(TH)- and VMAT2-positive synaptic varicosities. The presynaptic TH fluorescence level was also reduced. Patch-clamp experiments were performed in slices from 1-year-old mice to record unitary EPSCs (uEPSCs) of presumed cortical origin in the absence of G-protein-mediated modulation. In HD mice, the maximal amplitudes of uEPSCs amounted to 69% of the WT level which matches the loss of VGluT1+/SYP+ synaptic terminals in immunostained sections. These results identify impairment of cortico-striatal synaptic transmission and dopamine release as a potential basis of hypokinesia in HD.

Unitary, quantal and miniature GABA-activated synaptic chloride currents in cultured neurons from the rat superior colliculus.

The aim of this study was to identify the conductance change induced by one quantum of gamma-aminobutyric acid from axonal release sites on cultured superior colliculus neurons. Unitary (single cell-activated) inhibitory postsynaptic currents and spontaneous synaptic activity were recorded with patch clamp techniques in the whole cell configuration while superfusing the entire neuron with normal saline. Miniature inhibitory postsynaptic currents were recorded in the presence of tetrodotoxin and in reduced [Ca2+]o/[Mg2+]o. In addition, the membrane area contributing to synaptic activity was limited to a narrow window of 50 microns. Smaller neurons were chosen for recording to render a standard deviation of the "instrumental" noise of less than 1.5 pA at a holding voltage of -80 mV. After two weeks in vitro, the percentage of synaptically connected tectal neurons exceeded 50%. At holding voltages of -80 mV (Cl- equilibrium potential -12 mV) minimal amplitudes of unitary inhibitory postsynaptic currents were as low as 7-10 pA, while maximal amplitudes exceeded 500 pA. The mean time to peak and time constant of decay were 3.0 and 34.4 ms, respectively (n = 31). Fluctuating unitary inhibitory postsynaptic currents were deemed to be compound postsynaptic responses. Multiple Gaussian equations could be fitted to the amplitude histograms of unitary postsynaptic currents. This procedure rendered a quantal size between 5.0 and 10.9 pA (mean 7.1 pA; S.D. 1.78 pA) in five neurons from mature cultures. The amplitudes of statistically determined quantal inhibitory postsynaptic currents were slightly smaller than the independent estimate from somatic miniature inhibitory postsynaptic currents. The latter had a mean amplitude of 9.1 pA (S.D. 3.3 pA, n = 23), a mean time to peak of 1.65 ms (n = 9), and a mean time constant of decay of 16.2 ms (n = 9). Single channel recording from outside-out patches showed three to four main conductance states ranging from 9 to 22 pS. Single channel closures at the 21-24 pS level were occasionally observed during relaxation of miniature currents. The small size of whole cell quantal inhibitory postsynaptic currents and somatic miniature currents indicates that one GABA quantum opened only 5-15 single Cl- channels.

Interaction of calcium-permeable non-N-methyl-D-aspartate receptor channels with voltage-activated potassium and calcium currents in rat retinal ganglion cells in vitro.

Calcium-permeable non-N-methyl-D-aspartate receptor channels are now characterized in much detail, but still little is known about the consequences of Ca2+ influx through these channels in specific neuron types. We are interested in the role of Ca2+-permeable non-N-methyl-D-aspartate receptor channels during differentiation of retinal ganglion cells. However, in view of the conflicting data on the relative Ca2+ permeability of non-N-methyl-D-aspartate receptor channels in these neurons, a more systematic evaluation of permeation properties of different Na+ substitutes was necessary before proceeding with the main goal of the present study evaluating the effects of non-N-methyl-D-aspartate receptor activation on repetitive firing and voltage-activated K+ and Ca2+ conductances. Retinal ganglion cells were dissociated from the rat retina on postnatal day 5. They were selected by vital anti-Thy-1 immunostaining and repetitive firing behaviour and submitted to patch-clamp recording in the whole-cell configuration. Non-N-methyl-D-aspartate receptor channels were activated by application of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate. It was found that they were essentially impermeable to N-methyl-D-glucamine (P(NMDG)/P(Cs)<0.02), but not to choline (P(choline)/P(Cs)=0.24) and tetramethylammonium (P(TMA)/P(Cs)=0.23). When using N-methyl-D-glucamine as a substitute for Na+ to obtain bi-ionic conditions P(Ca)/P(Cs) varied between 0.08 to 1.40. Linear current voltage relation or little outward rectification corresponded to a low Ca2+ permeability (P(Ca)/P(Cs)=0.14). In about one third of the cells kainate-induced currents showed inward rectification and non-N-methyl-D-aspartate receptor agonists induced a substantially higher Ca2+ influx (P(Ca)/P(Cs)=0.64). Activation of non-N-methyl-D-aspartate receptors by kainate profoundly altered the repetitive discharge of retinal ganglion cells. In contrast to the continuously firing controls, cells generated only a few spikes at the beginning of a steady depolarization after kainate exposure. Among the candidates regulating the firing behaviour of retinal ganglion cells voltage-activated Ca2+ and K+ conductances were tested for their sensitivity to kainate application. It was found that even short conditioning pulses of kainate decreased the peak amplitudes of both voltage-activated K+ and voltage-activated Ca2+ currents. Only the latter effect required extracellular Ca2+ and was antagonized by increasing the intracellular Ca2+ buffering strength. Thus, suppression of calcium currents was induced by a non-N-methyl-D-aspartate receptor-mediated rise of the intracellular calcium concentration. The reduction of K+ currents did not depend on extracellular calcium and was insensitive to experimental manipulation of intracellular Ca2+ buffer strength. The interaction between Ca2+-permeable non-N-methyl-D-aspartate receptor channels and voltage-activated Ca2+ and K+ currents may represent an important regulatory mechanism to control the repetitive firing of developing retinal ganglion cells.

An abnormal response of retinoblastoma cells (Y-79) to neurotrophins.

PURPOSE: To clarify the expression of neurotrophins and their receptors in retinoblastoma (Rb) cells, to elucidate their potential role in the proliferation of neuroectodermal tumor cells, and to establish conditions for Rb cell differentiation. METHODS: The Rb-derived cell line Y-79 was grown in serum-free suspension or monolayer culture. Proliferating and differentiated cells were isolated and submitted to semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis, immunostaining, and flow cytometry. The proliferation rate of the cells was estimated by 5-bromo-2'-deoxyuridine (BrdU) incorporation, and the effects of neurotrophins and laminin on BrdU-incorporation, process outgrowth, or immunostaining were determined. RESULTS: In contrast to previously studied normal retinal precursor cells, Y-79 cells not only express nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and p75, but also the corresponding high affinity receptors TrkA, TrkB, and TrkC. Proliferation was stimulated by exogenous and endogenous neurotrophin receptor ligands. Inhibition of protein kinase phosphorylation with K252a blocked proliferation and promoted differentiation. The effect of K252a on differentiation was enhanced by the addition of soluble laminin. After 9 days of combined treatment, the fraction of differentiated cells amounted to 30%, differentiation being characterized by improved attachment, neurite outgrowth, expression of NF-68, and a loss of glial fibrillary acidic protein (GFAP) and parvalbumin immunoreactivity. These changes were accompanied by a downregulation of TrkB and TrkC, but not TrkA or p75. Differentiated cells were isolated and further grown in the absence of K252a. However, despite the high level of TrkA expression in differentiated cells, the addition of NGF had no effect on their survival. CONCLUSIONS: A mitogenic action of neurotrophins could contribute to retinal tumor growth.

Ligand- and voltage-gated ion channels are expressed by embryonic mouse retinal neurones.

The present study was intended to investigate whether voltage- and ligand-activated ion channels are expressed during prenatal development by neurones located in the ganglion cell layer of the mammalian retina. Whole cell patch clamp recordings from presumed mouse retinal ganglion cells revealed the expression of Na+, K+ and Ca2+ channels, predominantly of the low-voltage-activated type. Using local application of transmitter substances we further demonstrated that these cells are endowed with glutamate receptors of the N-methyl-D-aspartate (NMDA) and non-NMDA type as well as nicotinic acetylcholine, gamma-amino-butyric acid (GABA)A and glycine receptors. Voltage-gated conductances probably underlie spontaneous action potential generation by embryonic ganglion cells. The early expression of transmitter-gated ion channels indicates important functions of these channels in cell differentiation processes.

A readily releasable pool of single inhibitory boutons in culture.

The number of presynaptic vesicles that are immediately available for release, the readily releasable pool (RRP), is a strong determinant of synaptic strength and plasticity. The properties of the RRP in individual GABAergic synapses were examined in superior colliculus cultures. The RRP was depleted by high frequency trains and cumulative evoked IPSC amplitudes (CA) were calculated. The amplitude of monoquantal responses (q) was determined on the basis of mIPSC histograms. On average, the RRP, defined as CA/q, comprised about 10 vesicles. About 60% of the RRP could be released by a single stimulus. After depletion, the RRP was replenished with a time constant of about 14 s. These data provide information for further studies on the capacity of individual inhibitory synapses to modulate sensory information transfer.

Rapid genotyping of newborn gene mutant mice.

One important aspect of utilizing transgenic mice is the need to genotype them in order to distinguish mice that carry a disrupted gene or a transgene from mice that do not. Current methods for genotyping include isolation of genomic DNA from tail biopsies followed by PCR amplification. Particularly, both digestion of tail tissue using proteinase K as well as resuspension of purified DNA are time-consuming and were usually carried out overnight. Here, we describe a rapid and robust method for the genotyping of bdnf targeted mice which allows us to determine the genotype of newborn mice at the day of birth within 6 h. After a freezing-thawing step tail tissue is digested in less than 2 h, and the DNA is precipitated, resuspended and ready for PCR in about 60 min. The method could be easily adapted to a variety of different mutant mice and especially should benefit neuroscientists interested in using animals with known genotype very early in postnatal development.

In vitro identification of retinal ganglion cells in culture without the need of dye labeling.

We here describe a method for the identification of a distinct neuronal phenotype at all stages of development in culture without the need of any staining procedure. Based purely on a size criterion we can rapidly select vital retinal ganglion cells (RGCs) for further studies out of a mixed culture of rat retinal cells. In order to establish a size criterion for retinal cells of various age, RGCs were first labeled immunocytochemically with antibody against the ganglion cell-specific surface glycoprotein Thy-1. Soma diameters were then determined for labeled and unlabeled cells between embryonic day 16 (E16) and postnatal day 90 (P90). Unlabeled neurons of all ages had soma diameters between 3.6 microns and 12 microns (mean diameter: 6.3 microns). In contrast, soma diameters of RGCs ranged from 8.4 microns to 28 microns and the number of RGCs with large soma diameters increased with age. Thus, in a mixed retinal cell culture only RGCs are larger than 12 microns and can be selected solely based on their size. The validity of the size criterion during the whole period of retinal cell differentiation offers the possibility to study the development of cellular functions and ion channel properties in a distinct type of cell without the risk of artifacts introduced by staining.

Synaptic actions of tectofugal pathways on abducens motoneurons in the cat.

Organization of pathways between the superior colliculus (CS) and abducens motoneurons (VI-MNs) was studied in cats under pentobarbital anesthesia using intracellular recordings from VI-MNs and adjacent reticular neurons. Latencies of EPSPs elicited by contralateral CS stimulation indicate that a small fraction of the excitatory pathway may be monosynaptic while its major part is disynaptic. As suggested by an analysis of synaptic responses to microstimulation of the paramedian pontine region, excitatory impulses descend in the tectobulbospinal tract after crossing at midbrain levels. An attempt was made to identify interneurons of the excitatory tectoabducens pathway in the region just ventral and rostroventral to the VI-nucleus. About one-quarter of the reticular neurons in this region received monosynaptic excitation specifically from the contralateral CS. They were acceptable as interneurons with regard to other response characteristics too. Axonal projection to, or through, the abducens nucleus was demonstrated for some of them by intranuclear microstimulation or by tracing axons after Procion yellow injections. It is suggested that "premotor" interneurons of the excitatory tectoabducens pathway are concentrated in the vicinity of the abducens nucleic. A similar investigation of inhibitory responses to ipsilateral CS-stimulation indicates that inhibitory pathways are at least disynaptic and, for the most part, contain three or more synapses. In its initial trajectory the inhibitory pathway appears to be identical with the tectobulbospinal tract,but it decussates for the second time at caudal pontine levels to reach ipsilateral VI-MNs.

Separation of calcium currents in retinal ganglion cells from postnatal rat.

A culture system of the postnatal rat retina was established to investigate Ca2+ currents and synaptic transmission in identified neurons. Methods are described that allowed us to select retinal ganglion neurons (RGNs) in short term cultures (up to 48 h in vitro) and in long-term cultures (3 to 21 days in vitro). The specific aim of the present study was to identify channel specific components in whole-cell Ca2+ currents of RGNs and to clarify the potential use of the lanthanide Gd3+ as a selective Ca2+ channel blocker. About one third of freshly dissociated RGNs generated both low voltage activated Ca2+ currents (ICa(LVA)) and high voltage activated Ca2+ currents (ICa(HVA)). The remaining 2/3 or RGNs in short term culture and most RGNs in long-term culture displayed only ICa(HVA). The latter comprised at least three different components that were functionally rather similar, but could be separated pharmacologically. A significant portion (about 40%) of ICa(HVA) was irreversibly blocked by the N channel antagonist omega-CgTx (5 microM). The L channel antagonist nifedipine (10 microM) eliminated about 25% of ICa(HVA). Thus, about 1/3 of the HVA Ca2+ or Ba2+ current remained unaffected by either omega-CgTx or nifedipine. omega-AgaTx (200 nM) completely failed to block HVA Ca2+ or Ba2+ currents in RGNs. Gd3+ exerted contrasting actions on LVA and HVA Ca2+ currents. While ICa(LVA) consistently increased in the presence of Gd3+ (0.32-3.2 microM), ICa(HVA) always decreased, especially when using higher concentrations of Gd3+ (10-32 microM). The blocking action of Gd3+ was not restricted to the omega-CgTx-sensitive HVA current component, but also concerned omega-CgTx- and nifedipine-resistant components. The decay of Ca2+ currents was accelerated in the presence of Gd3+. Even in RGNs lacking ICa(LVA), application of 3.2 microM Gd3+ significantly reduced the time constant of decay from an average of 64 ms to 36 ms (voltage steps from -90 to 0 mV; 10 mM [Ca2+]o; 26 degrees C). This is in contrast to what had to be expected if an N-type HVA current component was selectively suppressed by Gd3+.Gd3+ diminished glutamatergic spontaneous synaptic activity in retinal cultures tested during the 3rd week in vitro. Both frequency and amplitude were reduced. Occasionally, the application was followed by a rebound increase of EPSC frequency. A stimulatory effect during application of Gd3+ has never been observed. These experiments indicate that RGNs express at least 4 different types of Ca2+ currents, that resemble in some aspects T, N and L channel currents.(ABSTRACT TRUNCATED AT 400 WORDS)

Glutamate-induced ionic currents in cultured neurons from the rat superior colliculus.

The ionic currents induced in cultured rat superior colliculus neurons by rapid application of glutamate (Glut) and the glutamate receptor agonists quisqualate (Quis) and N-methyl-D-aspartate (NMDA) were examined using the whole-cell patch clamp technique. Dissociated cell cultures consisting exclusively of superficial gray layer neurons from rats aged E21-P2 were used. After 7-10 days in vitro, all neurons responded to Glut and the selective agonists, NMDA and Quis. Glut was a mixed agonist, and a variable fraction (10-100%) of Glut-activated currents was due to involvement of NMDA receptors. The NMDA response was strongly regulated by extracellular Ca and Mg levels and modified by exposure to Quis. Quis transiently removed the block of NMDA-activated currents by D-amino-phosphonovaleric acid (APV).

Early onset of glutamatergic and GABAergic synaptic activity in the visual layers of the rodent superior colliculus.

During postnatal development, the retinocollicular pathway undergoes activity-dependent refinement, resulting in the precise retinotopic map seen in adults. Previous studies established that retinal efferents reach the mouse superior colliculus (SC) by embryonic day 16. Morphologically, synapses were found in the rat SC before birth. As part of an extended project aimed at understanding the development of synaptic transmission in the visual layers of the SC, we report here the presence of functionally active synapses immediately after birth. Circuit activity in mouse SC neurons was detected in horizontal slices of the visual layers using cell-attached voltage clamp. The spontaneous discharge of action potentials was abolished by glutamatergic blockers and facilitated by bicuculline, showing that circuit activity is based on synaptic transmission and that the action of gamma-aminobutyric acid is inhibitory. Using whole-cell voltage clamp, spontaneous glutamatergic postsynaptic currents as well as miniature GABAergic postsynaptic currents were recorded on postnatal day 1. Excitatory and inhibitory postsynaptic currents could also be evoked by electrical stimulation. Glutamatergic postsynaptic currents comprised both (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptor-mediated components. The early function of glutamatergic and GABAergic synaptic transmission in the visual layers of SC suggests that SC neurons are able to process information originating from retinal axons immediately after birth.

Glutamatergic and GABAergic synaptic currents in ganglion cells from isolated retinae of pigmented rats during postnatal development.

This study was aimed at characterizing the earliest phases of synaptogenesis in the mammalian retina. Spontaneous activity of ganglion cells in the isolated superfused retina was used as an indicator for the functionality of synaptic connections. Retinal ganglion neurons (RGNs) were identified by location of their somata in the ganglion cell layer (GCL) and by their ability to generate large (> 500 pA) voltage-activated sodium currents. Spontaneous spiking was found in many RGNs prior to cell perfusion. Between postnatal day (P) 1 and 18, a total of 195 RGNs was tested for light-induced currents, conductance changes in response to exogenous glutamate (Glu) and gamma-aminobutyric acid (GABA), and depolarizing or hyperpolarizing synaptic activity. The vast majority of the material was derived from RGNs at day P5. Whole-cell ion currents were always sampled at somatic sites, using either conventional or perforated patch whole-cell recordings. On day P5, 5% of tested RGNs (n = 73) were already responsive to light stimulation. A higher percentage of cells (23%, n = 187) generated spontaneous depolarizing currents that were regarded as glutamatergic excitatory postsynaptic currents (EPSCs), since (1) they were blocked by Glu antagonists, (2) they conformed to the Na+/Cs+ equilibrium potential, (3) and they displayed a time course characteristic of glutamatergic EPSCs. The mean EPSC amplitude was 19.0 pA (S.D. 11.83 pA). Amplitude distributions were fitted by multiple Gaussian equations rendering a quantal size of 6.6 to 9.1 pA at a holding voltage (Vh) of -70 mV (driving force about 70 mV). Spontaneous EPSCs were never observed under condition of Ca(2+)-free solutions, but they persisted in the presence of tetrodotoxin. Bath application of quisqualate (500 microM) consistently increased EPSC frequencies. In contrast to the relatively high percentage of RGNs generating spontaneous EPSCs, very few RGNs at P5 (3%, n = 187) displayed inhibitory postsynaptic currents (IPSCs), although by that time all tested RGNs (n = 14) were responsive to both exogenous Glu and GABA. These results indicate that in the postnatal rat retina development of excitatory synapses precedes the maturation of inhibitory afferents. Excitatory inputs to RGNs were to some extent functional before the animals opened their eyes. Glutamatergic synaptic activity may, thus, play an important role in shaping visual connections in the absence of visual experience.

Development of GABAergic synaptic connections in vivo and in cultures from the rat superior colliculus.

Synaptic activity in the superficial (i.e. visual) layer of the superior colliculus was investigated with intracellular microelectrodes using a preparation of the isolated superfused tectum from neonatal rat. It was found that by postnatal day 9 (i.e. before eye opening) the majority of neurons in the superficial gray layer (SGS, stratum griseum superficiale) were already capable of generating Cl(-)-dependent inhibitory postsynaptic potentials (IPSPs) in response to intracollicular stimulation. Properties and development of GABAergic synaptic connections were further characterized in a dissociated cell culture from the SGS. The cultures were prepared from E21 rat embryos and studied between 1 and 38 days in vitro (DIV). gamma-[3H]aminobutyric acid ([3H]GABA) uptake served to identify GABAergic neurons and to estimate their relative density. Axon terminals were labeled by indirect immunostaining for glutamic acid decarboxylase (GAD) and examined with light (LM) and electron microscopy (EM). Responsiveness to exogenous and endogenous GABA was investigated by recording ionic currents with patch clamp techniques. [3H]GABA uptake-positive neurons constituted about 40% of the whole cellular population dissociated from the SGS of E21 rats. After 2 weeks in culture, [3H]GABA uptake was observed in 45-60% of the cells with neuronal features. The relative number of GAD-immunoreactive neuronal perikarya ranged from 28 to 39%, after 2 weeks in vitro. Responsiveness to exogenous GABA was found in all freshly plated neurons. Release of GABA could be demonstrated after 2 DIV by recording spontaneous bicuculline-sensitive Cl- currents. These currents had the characteristics of GABAA receptor-mediated synaptic currents. However, even as late as DIV 6, very few vesicle-containing axonal terminals apposing postsynaptic specializations were revealed with EM. GAD-labeled puncta became clearly visible only after DIV 10-12. Between DIV 14 and 21, the intensity of immunostaining and the density of GAD-labeled synaptic contacts increased, reaching a maximum around DIV 28. GAD-positive puncta covered both neurons and non-neuronal cells. At the level of EM, GAD-positive terminals were shown to establish synaptic contacts with neuronal somata and processes, forming in the majority of cases (22 out of 32 stained terminals) symmetrical contacts. It is concluded that in the SGS of the rat superior colliculus GABAergic neurons and GABAA receptors are present before birth. In dissociated cell cultures ionic currents can be generated in response to endogenous GABA before axonal terminals of GABAergic neurons fully mature. Finally, our experiments show that visual activity is not a prerequisite for the formation of GABAergic synapses between neurons of the SGS.

Expression of depolarizing voltage- and transmitter-activated currents in neuronal precursor cells from the rat brain is preceded by a proton-activated sodium current.

The early expression of amiloride-sensitive proton-activated sodium currents (INa(H] was demonstrated using the giga-seal whole-cell voltage clamp technique in cells from the primordial tectum of E12 rat embryos. Less than 10% of these cells stained for tetanus toxin receptors after 2 h in vitro. However, after 10 h in vitro all cells with neuronal geometry were tetanus toxin-positive and capable of generating voltage-activated Na currents (INa(V] and high-voltage activated Ca2+-currents (ICa(HV]. INa(H) was expressed roughly in parallel with INa(V) and ICa(HV), but exceeded the former currents in amplitude by 50-100 times, reaching 600 pA and more. In 25% of the cells tested within the first 5 h in vitro INa(H) was, in fact, the only cationic inward current resolved. Responses to quisqualate and kainate appeared only after 3 days in vitro, and responses to N-methyl-D-aspartate/glycine were seen only after 4 days in vitro. These results suggest that the channels carrying INa(H) are present at the earliest stages of neuronal development.

Rat retinal ganglion cells express Ca(2+)-permeable non-NMDA glutamate receptors during the period of histogenetic cell death.

Local application of glutamate agonists to retinal ganglion cells (RGNs) was performed in retinae isolated from pigmented rats aged between 3 and 8 days postnatally. A vast majority of RGNs displayed current responses to glutamate (Glu), N-methyl-D-aspartate (NMDA), quisqualate (QA), alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoazolepropanoic acid (AMPA), kainate (KA) and domoate (DA). In Na(+)-free extracellular solution with elevated Ca2+, non-NMDA agonists elicited large (up to 200 pA) inward currents that were completely blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) and Cd2+. MK-801 also induced a partial block of cationic currents in Na(+)-free saline. In standard salt solutions, current-voltage relationships of Glu-R-mediated currents were often inwardly rectifying in the presence of D-aminophosphonovalerat (D-APV), as is typical of Ca(2+)-permeable non-NMDA receptors. The presence of inward rectification in the current voltage relationship was always associated with a high value of the cationic permeability ratio PCa2+/PCs+ (> 0.8). However, in about half of the investigated RGNs no inward rectification was observed under standard recording conditions. Our results lead to the suggestion that expression of Ca(2+)-permeable Glu receptor subunits may contribute to regulation of cell numbers in the postnatal retina.

Is GABA release modulated by presynaptic excitatory amino acid receptors?

The purely GABAergic nature of spontaneous synaptic activity in cultures from the neonatal rat superior colliculus (SC) is of great advantage in investigations aimed at characterizing presynaptic factors regulating GABAergic synaptic transmission. Using SC-derived cultures it was confirmed that excitatory amino acids (EAA) can induce a marked increase in the frequency of spontaneous synaptic Cl- currents (ICl(GABA)SYN). However, this tetrodotoxin-resistant facilitation of Ca2(+)-dependent GABA release required application of EEA to several neurons (multiple cell superfusion). In contrast, no frequency increase of Icl(GABA)SYN was seen with restricted access of EAA to only one neuron and the presynaptic axonal terminals (single cell superfusion). It is therefore concluded that the strong facilitatory effect of glutamate (Glu) and kainate (KA) on GABAergic synaptic activity, as observed under the condition of multiple cell superfusion, is mediated via somatodendritic excitatory amino acid receptors (EAARs).

Similarity and mutual exclusion of NMDA- and proton-activated transient Na+-currents in rat tectal neurons.

Vertebrate neurons respond to rapid elevation of [H+]o with transient Na+-selective currents (INa(H]. Since INa(H) and voltage-activated Ca2+-currents (ICa(V] are mutually exclusive and similarly affected by inorganic and organic Ca2+-blockers, it has been suggested that such a Na+-permeable state evolves from protonation of Ca2+-channels. We show here that in cultured neurons from embryonic rat superior colliculus N-methyl-D-aspartate (NMDA) provides conditions for generation of a current identical with INa(H), but without the requirement of an increase in free [H+]o. The transient NMDA-activated current (I(NMDA)T) is occluded by INa(H). Its time course is similar to that of INa(H). Both currents are inactivated by long exposure to high [H+]o. I(NMDA)T displays a linear current-voltage (I-V) relationship under conditions which cause a negative slope in the I-V relationship of the persistent NMDA-activated current (I(NMDA)P). This suggests that the biphasic response of tectal neurons to the glutamate-agonist NMDA results from superposition of two different currents.