Spike transmission and synchrony detection in networks of GABAergic interneurons.

The temporal pattern and relative timing of action potentials among neocortical neurons may carry important information. However, how cortical circuits detect or generate coherent activity remains unclear. Using paired recordings in rat neocortical slices, we found that the firing of fast-spiking cells can reflect the spiking pattern of single-axon pyramidal inputs. Moreover, this property allowed groups of fast-spiking cells interconnected by electrical and gamma-aminobutyric acid (GABA)-releasing (GABAergic) synapses to detect the relative timing of their excitatory inputs. These results indicate that networks of fast-spiking cells may play a role in the detection and promotion of synchronous activity within the neocortex.

Tonic activation of NMDA receptors by ambient glutamate enhances excitability of neurons.

Voltage clamp recordings and noise analysis from pyramidal cells in hippocampal slices indicate that N-methyl-D-aspartate (NMDA) receptors are tonically active. On the basis of the known concentration of glutamate in the extracellular fluid, this tonic action is likely caused by the ambient glutamate level. NMDA receptors are voltage-sensitive, thus background activation of these receptors imparts a regenerative electrical property to pyramidal cells, which facilitates the coupling between dendritic excitatory synaptic input and somatic action potential discharge in these neurons.

Different glutamate receptor channels mediate fast excitatory synaptic currents in inhibitory and excitatory cortical neurons.

Spontaneous excitatory postsynaptic currents (sEPSCs) and responses to rapid application of glutamate were recorded in excitatory spiny, pyramidal neurons and compared with those recorded in inhibitory aspiny interneurons. The sEPSC decay time constant was faster in aspiny interneurons (2.5 ms) compared with pyramidal neurons (4.6 ms). The decay time constant in response to a brief application (1 ms) of glutamate (10 mM) in patches excised from pyramidal and aspiny interneurons were similar (1.9 and 2.7 ms, respectively). However, the rate of desensitization was faster in patches from interneurons compared with pyramidal neurons (3.4 and 12.0 ms, respectively). In addition, single-channel conductance was larger in aspiny interneurons (27 pS) compared with pyramidal neurons (9 pS). These results indicate that pyramidal neurons and aspiny interneurons express different non-N-methyl-D-aspartate receptors and that selective desensitization of interneuron receptors may contribute to depression of inhibition.

Activation and desensitization of glutamate-activated channels mediating fast excitatory synaptic currents in the visual cortex.

Brief glutamate applications to membrane patches, excised from neurons in the rat visual cortex, were used to assess the role of desensitization in determining the AMPA/kainate receptor-mediated excitatory postsynaptic current (EPSC) time course. A brief (1 ms) application of glutamate (1-10 mM) produced a response that mimicked the time course of miniature EPSCs (mEPSCs). Direct evidence is presented that the rate of onset of desensitization is much slower than the decay rate of the response to a brief application of glutamate, implying that the decay of mEPSCs reflects channel closure into a state readily available for reactivation. Rapid application of glutamate combined with nonstationary variance analysis provided an estimate of the single-channel conductance and open probability, allowing an approximation of the number of available channels at a single synaptic site.

Mechanisms generating the time course of dual component excitatory synaptic currents recorded in hippocampal slices.

We studied with the whole-cell recording techniques, the mechanisms underlying the time course of the slow N-methyl-D-aspartate (NMDA), and fast non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) in hippocampal slices. The rising phase of the NMDA receptor-mediated component of the EPSC as well as the decaying phase of the NMDA and non-NMDA component were highly temperature-sensitive, suggesting that neither of these processes is determined by free diffusion of transmitter. Moreover, glutamate uptake blockers enhanced the responses to exogenously applied glutamate, but had no effect on the decay of either the NMDA or non-NMDA components of the EPSCs. On the other hand, open channel blockers known to modify NMDA channel kinetics reduced the EPSC decay time. Thus, the present results support a model in which the rise time and decay of the NMDA component are determined primarily by slow channel kinetics and the decay of the non-NMDA component is due either to channel kinetics or to desensitization.

Frequency-dependent synaptic depression and the balance of excitation and inhibition in the neocortex.

The stability of cortical neuron activity in vivo suggests that the firing rates of both excitatory and inhibitory neurons are dynamically adjusted. Using dual recordings from excitatory pyramidal neurons and inhibitory fast-spiking neurons in neocortical slices, we report that sustained activation by trains of several hundred presynaptic spikes resulted in much stronger depression of synaptic currents at excitatory synapses than at inhibitory ones. The steady-state synaptic depression was frequency dependent and reflected presynaptic function. These results suggest that inhibitory terminals of fast-spiking cells are better equipped to support prolonged transmitter release at a high frequency compared with excitatory ones. This difference in frequency-dependent depression could produce a relative increase in the impact of inhibition during periods of high global activity and promote the stability of cortical circuits.

Effects of cyclic GMP on the kinetics of the photocurrent in rods and in detached rod outer segments.

We investigated the effects of high concentrations of cytoplasmic cyclic GMP on the photocurrent kinetics and light sensitivity of the tiger salamander rod both in intact cells and in detached outer segments. Photoreceptors were internally perfused with cGMP by applying patch pipettes containing cGMP to the inner or outer segment. Large increases in the concentration of cGMP in the outer segment cytoplasm were achieved only when the patch pipette was applied directly to the outer segment. The dark-current amplitude increased with increasing cGMP concentrations up to approximately 1,400 pA. Internal perfusion with 5.0 mM cGMP introduced a delay of 1-3 s in the photocurrent. The magnitude of the delay was inversely proportional to the light intensity. In addition, the photocurrent time course was slowed down and the light sensitivity, measured 1 s after the flash, was decreased approximately 100-fold when compared with that of the intact cell. The observed effects of cGMP were compared with those predicted by a model that assumes that the initial photocurrent time course is determined by the kinetics of the light-activated phosphodiesterase (PDE) and the cGMP dependence of the light-sensitive channels. At high concentrations of cGMP, the experimental data were similar to those predicted by the model and based on the known biochemical properties of the light-activated PDE and cGMP-activated channels.

Excitatory synaptic currents in Purkinje cells.

The N-methyl-D-aspartate (NMDA) and non-NMDA classes of glutamate receptor combine in many regions of the central nervous system to form a dual-component excitatory postsynaptic current. Non-NMDA receptors mediate synaptic transmission at the resting potential, whereas NMDA receptors contribute during periods of postsynaptic depolarization and play a role in the generation of long-term synaptic potentiation. To investigate the receptor types underlying excitatory synaptic transmission in the cerebellum, we have recorded excitatory postsynaptic currents (EPSCS), by using whole-cell techniques, from Purkinje cells in adult rat cerebellar slices. Stimulation in the white matter or granule-cell layer resulted in an all-or-none synaptic current as a result of climbing-fibre activation. Stimulation in the molecular layer caused a graded synaptic current, as expected for activation of parallel fibres. When the parallel fibres were stimulated twice at an interval of 40 ms, the second EPSC was facilitated; similar paired-pulse stimulation of the climbing fibre resulted in a depression of the second EPSC. Both parallel-fibre and climbing-fibre responses exhibited linear current-voltage relations. At a holding potential of -40 mV or in the nominal absence of Mg2+ these synaptic responses were unaffected by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV), but were blocked by the non-NMDA receptor antagonist 6-cyano-2,3-dihydro-7-nitroquinoxalinedione (CNQX). NMDA applied to the bath failed to evoke an inward current, whereas aspartate or glutamate induced a substantial current; this current was, however, largely reduced by CNQX, indicating that non-NMDA receptors mediate this response. These results indicate that both types of excitatory input to adult Purkinje cells are mediated exclusively by glutamate receptors of the non-NMDA type, and that these cells entirely lack NMDA receptors.

Diversity of glutamate receptors in neocortical neurons: implications for synaptic plasticity.

The biochemical and functional characteristics of the AMPA subtype of the glutamate receptors expressed by pyramidal and non-pyramidal neurons of the neocortex have been studied in acute slices by means of single-cell RT-PCR and fast applications of glutamate on outside-out patches. Our results suggest that the predominant expression of the flop splice variants of the GluR1-4 AMPA subunits contributes to the faster desensitization of these receptors in non-pyramidal neurons compared to pyramidal cells where flip variants of GluR1-4 are dominant. Alternative splicing of AMPA receptors may therefore play an important role in regulating synaptic function in a cell-type specific manner.

The properties and function of inward rectification in rod photoreceptors of the tiger salamander.

1. Rod photoreceptors were isolated from the retinae of tiger salamanders and voltage clamped using the whole-cell patch-clamp technique. 2. Hyperpolarizing the cell to potentials more negative than -50 mV evoked an inward current termed Ih. 3. Ih did not turn on immediately following a hyperpolarizing step but showed a marked delay. The activation time course of Ih could be described by the sum of two exponential components of opposite polarity. 4. The steady-state chord-conductance was half activated at -67 mV. 5. The reversal potential of Ih was close to -30 mV in normal standard salt solution. Increasing the external potassium concentration tenfold shifted the reversal potential by +17 mV. 6. The conductance-voltage relation and the kinetic parameters were not affected by changes in the external potassium concentration. 7. When fully activated, the zero-current conductance underlying Ih depended on the square root of the concentration of external potassium. 8. The permeability ratio PNa/PK depended on the external potassium concentration. It was 0.2 at an external potassium concentration of 2.0 mM and 0.3 at an external potassium concentration of 10.0 mM. The interaction of potassium with Ih suggests that Ih is a multi-ion pore. 9. It is concluded that Ih differs from the inward rectifier that is found in egg cells, frog muscle and heart muscle. 10. The kinetics and voltage sensitivity of Ih suggest that it does not play a role in the dark resting state or in the response to dim flashes of light. Its properties indicate that it may have a major role in the response to bright flashes.

Developmental regulation of NMDA receptor-mediated synaptic currents at a central synapse.

The central nervous system has extraordinary plasticity in early life. This is thought to involve N-methyl-D-aspartate (NMDA) receptors which, along with the non-NMDA receptors, mediate fast excitatory synaptic transmission. Although NMDA receptors may be transiently enhanced early in life, it has not been possible to demonstrate directly a functional change in the NMDA receptor-mediated synaptic response because of the voltage-dependence of the NMDA conductance and the overlapping inhibitory synaptic conductances. Here I report that the duration of evoked NMDA-receptor-mediated excitatory postsynaptic currents (e.p.s.cs) in the superior colliculus is several times longer at early developmental stages compared to that measured in older animals. In contrast, the amplitude of NMDA-receptor-mediated miniature e.p.s.cs does not change during development. The kinetic response of excised membrane patches to a brief activation of NMDA receptors is similar to that of the NMDA e.p.s.c, which suggests that the time course of the NMDA e.p.s.c. in the superior colliculus reflects slow NMDA channel properties as in the hippocampus. Therefore, these data indicate that the molecular properties of NMDA receptors are developmentally regulated and thus may be controlling the ability of synapses to change in early life.

The interaction of potassium with the activation of anomalous rectification in frog muscle membrane.

1. Inward rectification of frog muscle membrane was analysed with the Vaseline gap method. 2. Hyperpolarization, under voltage clamp, produced inward potassium currents, which had a component that activated with a time constant, tau K. 3. The activation time constant tau K of the inward potassium currents was voltage dependent. For a given external potassium concentration, the time constant was maximal for potentials near the potassium equilibrium potential, EK. 4. The potassium chord conductance gK, had a sigmoidal voltage dependency, increasing initially e-fold per 11.6 mV of hyperpolarization. 5. When the internal potassium concentration was fixed, raising external potassium induced a shift of the tau K-V and the gK-V relations in the positive direction along the voltage axis. That shift was comparable to the change in EK. 6. No shift of the tau K-V and the gK-V relations was observed when the internal potassium was reduced from 150 to 50 mM. 7. Changes of internal sodium concentration between 5 and 100 mM did not significantly effect the magnitude of inward rectification.

Electrical synapses between GABA-releasing interneurons.

Although gap junctions were first demonstrated in the mammalian brain about 30 years ago, the distribution and role of electrical synapses have remained elusive. A series of recent reports has demonstrated that inhibitory interneurons in the cerebral cortex, thalamus, striatum and cerebellum are extensively interconnected by electrical synapses. Investigators have used paired recordings to reveal directly the presence of electrical synapses among identified cell types. These studies indicate that electrical coupling is a fundamental feature of local inhibitory circuits and suggest that electrical synapses define functionally diverse networks of GABA-releasing interneurons. Here, we discuss these results, their possible functional significance and the insights into neuronal circuit organization that have emerged from them.

Properties of GABAA receptors underlying inhibitory synaptic currents in neocortical pyramidal neurons.

Rapid applications of GABA (from 10 microM to 10 mM) to outside-out patches were used to study the role that the kinetic properties of GABAA receptors play in determining the time course of IPSCs in neocortical pyramidal neurons. Currents induced by rapid applications of brief (1 msec) pulses of GABA (1 mM) showed a biexponential decay phase that seems to involve the entry of GABAA receptors into desensitized states. This conclusion is based on the similar fast decay kinetics of the response to brief and prolonged pulses of GABA and on the correlation between the degree of paired-pulse depression and the decay rate of the currents induced by brief pulses. Under nonequilibrium conditions we found that the concentration-response curve of pyramidal GABAA receptors has an EC50 of 185 microM (GABA pulse of 1 msec). The decay time course of the patch currents in response to brief applications of GABA was insensitive to agonist concentrations at the range from 50 microM to 10 mM. Faster decay rates were observed only in response to pulses of 10 microM GABA. These data are compatible with the suggestion that briefer openings derive from a monoliganded state and that these are negligible when receptor activation is >2%. Assuming that GABA transients at neocortical synapses are fast, a several millimolar GABA concentration would be needed to saturate the postsynaptic GABAA receptors.

Developmental regulation of basket/stellate cell-->Purkinje cell synapses in the cerebellum.

We used paired recordings to study the development of synaptic transmission between inhibitory interneurons of the molecular layer and Purkinje cells in the cerebellar cortex of the rat. The electrophysiological data were combined with a morphological study of the recorded cells using biocytin or Lucifer yellow staining. Thirty-one interneuron-Purkinje cell pairs were obtained, and 11 of them were recovered morphologically. The age of the rats ranged from 11 to 31 d after birth. During this period synaptic maturation resulted in an 11-fold decrease in the average current evoked in a Purkinje cell by a spike in a presynaptic interneuron. Unitary IPSCs in younger animals exhibited paired-pulse depression, whereas paired-pulse facilitation was found in more mature animals. These data suggest that reduction in transmitter release probability contributed to the developmental decrease of unitary IPSCs. However, additional mechanisms at both presynaptic and postsynaptic loci should also be considered. The decrease of the average synaptic current evoked in a Purkinje cell by an action potential in a single interneuron suggests that as development proceeds interneuron activities must be coordinated to inhibit efficiently Purkinje cells.

Burst firing induces a rebound of synaptic strength at unitary neocortical synapses.

High-frequency activity produces transient depression at many synapses but also, as recently demonstrated, may accelerate the recovery from use-dependent depression. We have examined the possible consequences of this synaptic mechanism in neocortical excitatory synapses by recording simultaneously from presynaptic pyramidal neurons and their postsynaptic targets. Brief bursts of high-frequency spikes produced a strong depression of the amplitude of unitary excitatory postsynaptic currents (uEPSCs). However, when burst firing was combined with low-frequency ongoing activity, we found that the strong synaptic depression was followed by a transient rebound of synaptic strength. This rebound overshot the low-frequency baseline values and lasted 1-2 s. These results suggest that in the presence of ongoing activity, neocortical synapses may functionally facilitate following burst firing.

A network of fast-spiking cells in the neocortex connected by electrical synapses.

Encoding of information in the cortex is thought to depend on synchronous firing of cortical neurons. Inhibitory neurons are known to be critical in the coordination of cortical activity, but how interaction among inhibitory cells promotes synchrony is not well understood. To address this issue directly, we have recorded simultaneously from pairs of fast-spiking (FS) cells, a type of gamma-aminobutyric acid (GABA)-containing neocortical interneuron. Here we report a high occurrence of electrical coupling among FS cells. Electrical synapses were not found among pyramidal neurons or between FS cells and other cortical cells. Some FS cells were interconnected by both electrical and GABAergic synapses. We show that communication through electrical synapses allows excitatory signalling among inhibitory cells and promotes their synchronous spiking. These results indicate that electrical synapses establish a network of fast-spiking cells in the neocortex which may play a key role in coordinating cortical activity.

Activation kinetics of retinal cones and rods: response to intense flashes of light.

Cone photoreceptors are less sensitive to light and the duration of their photoresponse is shorter than that of rods. In salamander rods and cones, we identified 3 components in membrane currents activated by bright flashes of light: an early receptor current (ERC) resulting from charge displacement within visual pigments, a saturation photocurrent generated by the closure of the cGMP-sensitive channels, and a putative Na-Ca exchanger current. The time courses of both the ERC and the onset of the saturation photocurrent were similar in rods and cones. The putative Na-Ca exchanger current, on the other hand, is 4- to 8-fold faster in cones. The onset of the saturation photocurrent consisted of a delay followed by a fast relaxation with an exponential time course. In both photoreceptor types the delay and the time course of the fast relaxation are dependent on light intensity and reach a limiting value when about 1% of the photopigment is bleached. The limiting value of the delay, about 8 msec, and of the relaxation time constant, about 2 msec, are nearly identical in rods and cones. The near identity of these parameters implies that at least 2 kinetic steps in the activation response of rods and cones are quantitatively similar. These findings suggest that the functional differences between rods and cones may arise from disparities in the processes that restore the components of the phototransduction cascade to their dark level and not from differences in the activation processes.

Voltage-activated potassium channels in the plasma membrane of rod outer segments: a possible effect of enzymatic cell dissociation.

Using patch-clamp techniques, we recorded single-channel currents from the plasma membrane of the outer segment of isolated light-adapted rods. The channels are potassium-selective and their conductance is about 87 pS. The channels are activated by depolarization and are not sensitive to cytoplasmic calcium, they are exclusively found in rods isolated with the proteolytic enzyme papain, and are not detected in rods isolated by mechanical means. Thus, these channels do not exist in an activatable form in the outer segment plasma membrane under physiological conditions. The channels might be derived from a normally inaccessible structure, such as the disk membrane, or, alternatively, they might be a modified form of a channel that is not active in the intact rod.

Morphology and physiology of cortical neurons in layer I.

The electrophysiological and morphological properties of layer I neurons were studied in visual cortex slices from 7- to 19-d-old rats using whole-cell recording and biocytin labeling. A heterogeneous population of small, nonpyramidal neurons was found. Approximately one third of the cells we recorded were neurogliaform cells; another third were multipolar neurons with axons descending out of layer I. The remaining cells were heterogeneous and were not classified. In slices from 7- to 10-d-old animals only, we identified Cajal-Retzius cells. Neurogliaform neurons had a very dense local axonal field, which was largely contained within layer I. Cells with descending axons had a relatively sparse local axonal arbor and projected at least to layer II and sometimes deeper. Spiking in neurogliaform neurons was followed by an afterdepolarizing potential, whereas spiking in cells with descending axons was followed by a slow after-hyperpolarizing potential (AHP). In addition, neurogliaform cells exhibited less spike broadening and a larger fast AHP after single spikes than did cells with descending axons. Generally, cells in layer I received synaptic inputs characterized as either GABA- or glutamate-mediated, suggesting the presence of excitatory and inhibitory inputs. With their output largely limited to layer I, neurogliaform cells could synapse with other layer I neurons, the most distal dendritic branches of pyramidal cells, or the dendrites of layer II/III interneurons, which invade layer I. Cells with descending axons could contact a wide variety of cortical cells throughout their vertical projection.