Serotonin regulates gap junction coupling in the developing rat somatosensory cortex.

To further elucidate the role of the neuromodulatory transmitter serotonin (5-HT) during early postnatal development of the neocortex, we investigated the effects of 5-HT on gap junction coupling in the somatosensory cortex of rats aged between postnatal days 7 and 10. The gap junction-permeable tracer neurobiotin was injected into single neurons via microelectrodes or patch pipettes. Under control conditions, clusters of about 25 tracer-coupled neurons were observed. Serotonin reduced dye-coupling between lamina II/III pyramidal cells in a concentration-dependent and reversible manner. The 1,4,5-inositol triphosphate (IP3) receptor antagonist heparin as well as the protein kinase C inhibitor NPC 15437 suppressed the uncoupling action of 5-HT, suggesting that the serotonergic effect involved IP3 receptor-mediated release of calcium ions from intracellular stores. In contrast, the 5-HT-induced reduction in gap junction coupling was not antagonized by Rp-adenosine-3',5'-cyclic monophosphothionate, an inhibitor of cAMP dependent protein kinase. The uncoupling effect of 5-HT was mimicked by 5-HT2 receptor agonists and antagonized by the 5-HT2 receptor antagonist ritanserin, indicating that 5-HT suppressed gap junction coupling via activation of 5-HT2 class receptors. Our results suggest that the developmental functions of 5-HT not only involve the modulation of chemical synaptic transmission but also include the regulation of the gap junctional communication system during differentiation of the neocortex.

Regulation of gap junction coupling in the developing neocortex.

In the developing mammalian, neocortex gap junctions represent a transient, metabolic, and electrical communication system. These gap junctions may play a crucial role during the formation and refinement of neocortical synaptic circuitries. This article focuses on two major points. First, the influence of gap junctions on electrotonic cell properties will be considered. Both the time-course and the amplitude of synaptic potentials depend, inter alia, on the integration capabilities of the postsynaptic neurons. These capabilities are, to a considerable extent, determined by the electrotonic characteristics of the postsynaptic cell. As a consequence, the efficacy of chemical synaptic inputs may be crucially affected by the presence of gap junctions. The second major topic is the regulation of gap junctional communication by neurotransmitters via second messenger pathways. The monoaminergic neuromodulators dopamine, noradrenaline, and serotonin reduce gap junction coupling via activation of two different intracellular signaling cascades--the cAMP/protein kinase A pathway and the IP3/Ca2+/protein kinase C pathway, respectively. In addition, gap junctional communication seems to be modulated by the nitric oxide (NO)/cGMP system. Since NO production can be stimulated by glutamate-induced calcium influx, the NO/cGMP-dependent modulation of gap junctions might represent a functional link between developing glutamatergic synaptic transmission and the gap junctional network. Thus, it might be of particular importance in view of a role of gap junctions during the process of circuit formation.

Nitric oxide-stimulated increase in intracellular cGMP modulates gap junction coupling in rat neocortex.

In the present study we demonstrate that gap junction coupling between developing layer II/III pyramidal cells in rat sensorimotor cortex is strongly modified by the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) system. Dye coupling was revealed by intracellular injection of the gap junction-permeable tracer neurobiotin into single neurones. Following incubation of slices with sodium nitroprusside, a source of NO, the size of dye-coupled cell clusters was significantly reduced. In many cases, 2-3 cells remained strongly dye-coupled. These effects were blocked by intracellular injection of the guanylyl cyclase inhibitor cystamine and mimicked by both application of the membrane-permeant cGMP analogue 8-Br-cGMP and intracellular injection of cGMP. cGMP injection also induced a 60% increase in neuronal input resistance. These results indicate that NO modulates gap junction coupling in the developing neocortex via stimulation of guanylyl cyclase.

Intracellular acidification reduced gap junction coupling between immature rat neocortical pyramidal neurones.

1. Developmental changes in electrophysiological properties of pyramidal neurones correlated with the developmental decline in gap junction-dependent dye coupling were investigated in coronal slices of rat prefrontal and sensorimotor cortex. Effects of intracellular acidification induced by application of weak organic acids on neuronal dye coupling, electrotonic parameters as well as synaptic potentials were examined using the patch clamp technique. Optical monitoring of intracellular pH revealed an acidic shift of 0.4-0.5 pH units following sodium propionate application. 2. Dye coupling between layer II-III neurones was prominent during the first two postnatal weeks. During this period, pre-incubation of slices with 30 mM of the sodium salts of weak organic acids reduced the number of cells coupled to the injected neurones by 64%. 3. Between postnatal days 1 and 18, the mean neuronal input resistance decreased significantly (by 81.0%). Both the membrane time constant (tau 0) and the first equalizing time constant (tau 1) also showed a significant developmental decline of 25.8 and 65.8%, respectively. Electrotonic length decreased by 34.9%. The electrophysiological properties of neurones displayed a pronounced intercellular variability which decreased with on-going development. 4. During the first two postnatal weeks, intracellular acidification led to a mean increase in neuronal input resistance of 55.9% and a mean decreae in electrotonic length of 22.2%. The membrane time constant was reduced by approximately 25% in the majority of neurones tested. Significant electrophysiological effects induced by intracellular acidification were not detected in uncoupled neurones from 18-day-old rats. 5. EPSP width at half-maximal amplitude showed a substantial reduction of approximately 50%, while rise times of the non-NMDA receptor-mediated EPSP components displayed no significant change during development. Both weak organic acids, as well as the gap junction blocker 1-octanol, reduced excitatory synaptic transmission independent of developmental age. 6. We conclude that gap junction permeability is regulated by intracellular pH in developing layer II-III pyramidal cells in the rat neocortex. The prominent correlation between pH-induced reduction in dye coupling and changes in electrophysiological cell properties suggests a significant influence of gap junctions on synaptic integration and information transfer in the immature neocortex.

Dye coupling between pyramidal neurons in developing rat prefrontal and frontal cortex is reduced by protein kinase A activation and dopamine.

During early postnatal development, lamina II/III pyramidal cells in rat neocortex are extensively coupled via gap junctions. The factors regulating gap junction permeability, as well as the mechanisms underlying the developmental uncoupling process are not understood. To investigate the influence of protein kinase A-mediated phosphorylation on dye coupling in the developing neocortex, pyramidal cells in slices of rat frontal and prefrontal cortex were injected intracellularly with the tracer neurobiotin. Control injections revealed clusters of about 30 dye-coupled neurons. Preincubation with forskolin or direct activation of protein kinase A with Sp-cAMPS reduced the number of coupled cells by about 70%. A significant reduction in dye coupling was also observed following incubation with dopamine. Application of receptor selective agonists and antagonists revealed that the uncoupling was mediated by both dopamine D1 and D2 receptors. The protein kinase A inhibitor Rp-cAMPS reduced the effect of dopamine, suggesting that the neurotransmitter regulates gap junction permeability via protein kinase A activation. In the presence of either forskolin, Sp-cAMPS, or dopamine, neurons displayed a significantly higher input resistance compared to control conditions. During the second postnatal week, transient application of forskolin to single neurons reversibly increased input resistance. At later developmental stages when coupling incidence had declined, this action of forskolin was no longer observed. Our data demonstrate a dependence of gap junction permeability on protein kinase A activity and on dopamine receptor activation in developing rat neocortical neurons. These mechanisms may modulate junctional permeability during the period of circuit formation.

Beta-adrenoreceptor activation reduces dye-coupling between immature rat neocortical neurones.

The present study examined the effect of activation of adrenergic receptors by noradrenaline and the beta-receptor selective agonist isoproterenol on dye-coupling between developing lamina II/III pyramidal neurones in rat prefrontal and sensorimotor cortex. To assess dye-coupling neurones were intracellularly injected with neurobiotin. Under control conditions injections performed in slices obtained from neonatal rats (7-10 postnatal days) resulted in clusters of more than 30 tracer-coupled neurones. Preincubation with either isoproterenol or noradrenaline reduced the number of coupled cells by 60-80%. The effect of isoproterenol was suppressed by the beta 1-adrenoreceptor antagonist atenolol. Our results indicate that modulation of gap junction coupling between differentiating neocortical neurones might be one important function of noradrenergic afferents during early postnatal development of the neocortex.

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.

Long-range horizontal connections between supragranular pyramidal cells in the extrastriate visual cortex of the rat.

In this study, we examined the morphological structure and synaptic physiology of long-range axon projections among supragranular pyramidal cells in the extrastriate visual cortex of the rat. Intra- and extracellular recordings from layer II/III pyramidal cells were performed in brain slices of area 18a following extracellular stimulation of either the underlying white matter or within layer II/III. Neurons were injected with biocytin for two-dimensional reconstruction of their axon arborizations. The conduction velocity of afferent fibers (0.58 m/s) was twice as high as that of intracortical tangential fibers (0.28 m/s). Layer II/III cells were mainly di- or polysynaptically driven by afferent activation, but predominantly monosynaptically driven from intracortical stimulation sites. The afferent as well as intracortically evoked postsynaptic potentials showed a very similar time course and shape. From both stimulation sites, suprathreshold action potentials could be elicited. The current threshold for a postsynaptic response and the slope and width of excitatory postsynaptic potentials (EPSPs) increased with the distance of lateral stimulation. The morphological properties of layer II/III pyramidal cell axon collaterals closely corresponded to the electrophysiological results. Long-range intraareal axon collaterals could be followed up to 1 mm within the supragranular layers. Their length-distance distribution showed an inverse relationship to the threshold currents of EPSPs. Pyramidal cells exhibited regularly spaced patches of horizontal axon collaterals with an interpatch distance of about 250 microns. We concluded that the supragranular horizontal network in the extrastriate visual cortex of the rat is qualitatively very similar to that of cats and monkeys. However, quantitative differences exist in its spatial extent and physiological characteristics.

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.

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.