Arthur Konnerth

Developmental profile and synaptic origin of early network oscillations in the CA1 region of rat neonatal hippocampus

1 By applying fura‐2‐based fluorometric calcium imaging to neonatal rat hippocampal slices we identified a developmentally regulated spontaneous neuronal activity in the CA1 region of the hippocampus. The activity consisted of bursts of intracellular Ca2+ transients recurring synchronously at a slow rate of 0.4–2 min−1 in the entire population of pyramidal neurones and interneurones. 2 These early network oscillations (ENOs) were present during a restricted period of postnatal development. Thus, they were not detected at the day of birth (P0), at P1–P4 they consisted of bursts of large (up to 1.5 μm) Ca2+ transients, gradually transforming into regularly occurring, smaller Ca2+ transients during the subsequent week. Beyond P15–P16 no ENOs were detected. 3 The ENOs were blocked by tetrodotoxin (TTX) and by a reduction in temperature from 33–35°C to 20–22°C. By combining fluorometric imaging with whole‐cell current‐clamp recordings, we found that each ENO‐related Ca2+ transient was associated with a high‐frequency (up to 100 Hz) train of action potentials riding on a depolarizing wave. 4 Recordings in the voltage‐clamp mode revealed barrages of synaptic currents that were strictly correlated with the ENO‐associated Ca2+ transients in neighbouring pyramidal neurones. Perfusing the cells with an intracellular solution that allowed for a discrimination between GABAA and glutamate receptor‐mediated currents showed that these barrages of synaptic currents were predominantly of GABAergic origin. 5 The ENOs were totally blocked by the GABAA receptor antagonist bicuculline and they were also substantially reduced by the glutamatergic antagonists d,l‐2‐amino‐5‐phosphonovaleric acid (d,l‐APV) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX). 6 Synaptic stimulation and application of the GABAA receptor agonist muscimol mimicked the spontaneous Ca2+ transients in pyramidal neurones. The efficacy of muscimol in evoking Ca2+ transients decreased during development in parallel with the gradual disappearance of the ENOs. 7 The developmental decrease in the amplitude of ENO‐associated Ca2+ transients occurred in parallel with the transformation of the excitatory synaptic transmission in the hippocampus from the immature GABAergic to the mature glutamatergic form. Thus, at the beginning of the first postnatal week single‐shock synaptic stimulation produced Ca2+ transients that were completely blocked by bicuculline. At the end of the second postnatal week the same type of evoked synaptic stimulation produced a Ca2+ transient that was little affected by bicuculline but was abolished by the combined application of d,l‐APV and CNQX. 8 These results demonstrate the presence of periodic and spontaneous Ca2+ transients in the majority of pyramidal cells and interneurones of the neonatal CA1 hippocampal network. These ENOs exhibit a highly region‐specific developmental profile and may control the activity‐dependent wiring of the synaptic connectivity during early postnatal development.

Ryanodine receptor-mediated intracellular calcium release in rat cerebellar Purkinje neurones.

1. Ryanodine receptor‐mediated Ca2+ release was investigated in Purkinje neurones of rat cerebellar slices by using whole‐cell patch‐clamp recordings combined with fluorometric digital imaging of cytoplasmic Ca2+ concentration ([Ca2+]i). 2. Caffeine caused a transient increase in [Ca2+]i in the somata and dendrites of Purkinje neurones. Caffeine‐induced Ca2+ transients were not associated with a membrane inward current and persisted in Ca(2+)‐free external solutions, indicating that they are caused by Ca2+ released from intracellular stores. The amplitudes of the caffeine‐mediated elevations in [Ca2+]i were strongly dependent on the baseline level of [Ca2+]i. 3. Intracellular application of Ruthenium Red through the patch pipette blocked caffeine‐induced Ca2+ transients in Purkinje neurones. Ryanodine when applied either intra‐ or extracellularly caused a use‐dependent block of caffeine‐induced Ca2+ release. 4. Depolarization‐induced Ca2+ transients were strongly prolonged by caffeine. Several lines of evidence suggest that these prolongations reflect Ca(2+)‐induced Ca2+ release. 5. Despite the presence of skeletal muscle type ryanodine receptors in Purkinje neurones, depolarizing pulses failed to induce any changes in [Ca2+]i when the influx of Ca2+ through voltage‐gated channels was prevented by using Ca(2+)‐free solution, or when applying blockers of voltage‐gated Ca2+ channels. 6. Dendritic Ca2+ transients produced by stimulation of the excitatory climbing fibre synaptic input were also prolonged by caffeine, indicating that ryanodine receptor‐mediated release of Ca2+ may be involved in synaptic signalling in cerebellar Purkinje neurones. 7. Ryanodine receptor‐mediated release of Ca2+ in cerebellar Purkinje neurones can be explained by a model in which release of Ca2+ is strongly facilitated by the co‐operative action of Ca2+, caffeine and/or ryanodine. Our results suggest that Ca2+ release in these central neurones becomes prominent only during episodes of intensive electrical activity associated with increased Ca2+ entry.

Early postnatal switch in magnesium sensitivity of NMDA receptors in rat CA1 pyramidal cells

1 Whole‐cell patch‐clamp recordings of iontophoretically induced N‐methyl‐D‐aspartate (NMDA) receptor‐mediated currents (INMDA) in CA1 pyramidal cells in hippocampal slices from 1‐ to 40‐day‐old rats were used to characterize developmental changes in the Mg2+ sensitivity of NMDA receptors. 2 The dose‐response relations for extracellular Mg2+ blockade of INMDA indicated a high affinity binding of Mg2+ to NMDA receptors at membrane potentials more negative than −60 mV, independent of postnatal age. 3 Depolarizing the cells unblocked NMDA receptors by decreasing their affinity for Mg2+. The efficacy of depolarization in unblocking NMDA receptors markedly increased after postnatal day 4 (P4), endowing the receptors with a greater voltage dependence. 4 The NR2B subunit‐specific NMDA antagonist ifenprodil reduced INMDA in pyramidal cells of all ages. The sensitivity of INMDA to ifenprodil was greatest during the first postnatal week and decreased thereafter, indicating an enhanced contribution of NR2B subunit‐containing NMDA receptors to INMDA in the first week after birth. 5 In the first postnatal week, the ifenprodil‐insensitive INMDA component had a lower voltage dependence than the total INMDA. In older pyramidal cells, the voltage dependence of the ifenprodil‐insensitive component and the total INMDA were similar. 6 In another set of CA1 pyramidal cells, single‐cell reverse transcription and polymerase chain reaction (RT‐PCR) were used to characterize concomitant developmental changes in NMDA subunit mRNA expression. The mRNA for the NR2D subunit was detected during the first postnatal week in 50% of the cells and disappeared thereafter. The proportion of cells expressing the NR2A and NR2B subunits remained relatively constant throughout the first five postnatal weeks. 7 We conclude that NMDA receptors in hippocampal CA1 pyramidal cells are effectively blocked by Mg2+ at all ages. After 4 days they become much less sensitive to Mg2+ at depolarized membrane potentials. This postnatal switch in voltage control of Mg2+ binding to NMDA receptors may be due to the downregulation of NR2D subunit expression in developing CA1 pyramidal cells.

Axonal calcium entry during fast ‘sodium’ action potentials in rat cerebellar Purkinje neurones.

1. Using laser‐scanning confocal microscopy, fast Ca2+ transients were recorded in individual not yet myelinated axons of Purkinje neurones in cerebellar slices from young rats. Axonal Ca2+ transients could be detected during a single action potential and had progressively larger amplitudes when the number of action potentials was increased. 2. Under voltage‐clamp conditions, axonal Ca2+ transients were as large as those observed in dendrites and in the cell body. Axonal Ca2+ transients were completely blocked by 100 nM of the neurotoxin omega‐agatoxin IVA, indicating that they were caused by Ca2+ entry through P‐type voltage‐gated Ca2+ channels. 3. In conclusion, our results demonstrate action potential‐mediated Ca2+ entry through voltage‐gated Ca2+ channels in axons of cerebellar Purkinje neurones. Experimental evidence indicates that the resulting transient Ca2+ accumulations regulate the frequency of action potentials travelling along the axon.

Dendritic signal integration.

Recent studies have identified various forms of active dendritic signals that may contribute to neuronal integration. One of the most remarkable findings is the demonstration of highly localized Ca2+ transients that are limited to small dendritic segments and even to single dendritic spines. In addition, through use of the powerful two-photon excitation imaging technique, it has been possible to reveal the existence of dendritic Ca2+ signals under in vivo conditions. Finally, active backpropagation of action potentials into dendrites has been shown to boost dendritic Ca2+ signals supralinearly and, thus, to contribute to the induction of long-term potentiation.

Localized calcium signalling and neuronal integration in cerebellar Purkinje neurones

The use of high resolution imaging techniques has revealed new forms of dendritic signal integration in neurones. In contrast to electrical signals that have a more widespread influence on the cell, brief Ca2+ transients resulting from synaptic activation are often restricted to a small part of the dendritic tree. In cerebellar Purkinje neurones, different levels of Ca2+ signalling have been observed that may involve the entire neurone or be spatially limited to fine dendritic structures. The Ca2+ signals accompanying subthreshold excitatory postsynaptic potentials resulting from stimulation of the excitatory parallel fibre input can be restricted to regions as small as a spiny dendrite or a single dendritic spine. With the recruitment of increasing numbers of inputs there is a summation of Ca2+ signals in highly restricted regions of the spiny dendrites that is independent of electrical summation at the soma. Of a number of potential sources that could provide the Ca2+ responsible for the postsynaptic changes, Ca2+ entry through voltage-gated Ca2+ channels has received the most support, although other sources like Ca2+ entry through ligand-gated channels and especially Ca2+ release from intracellular stores need to be considered.

Molecular determinants of NMDA receptor function in GABAergic neurones of rat forebrain.

1. The functional and molecular properties of NMDA receptors (NMDA‐Rs) were studied in single, visually identified GABAergic medial septal neurones of the rat forebrain using patch clamp, fluorometric Ca2+ measurements and the single‐cell reverse transcription‐polymerase chain reaction (RT‐PCR) technique. 2. Large neurones close to the mid‐line of the medial septal region were shown by the expression of mRNA for a form of glutamate decarboxylase (GAD65) to be almost exclusively GABAergic. A variety of NR2 subunit combinations were detected in the same population of neurones. When tested for NR2A‐C, all but one neurone were shown to express mRNA for NR2B. The NR2B subunit mRNA was usually detected together with NR2A or NR2C. mRNA for NR2D was detected in most neurones from a separate batch of cells tested only for this subunit. 3. Single channel measurements in outside‐out patches combined with RT‐PCR on the same cell showed that NMDA‐R channels from these neurones had main single channel conductance levels of 42 pS in 2 mM Ca2+ and 49 pS in 1 mM Ca2+. In addition, a number of other conductance levels were observed, with values in 2 mM Ca2+ of 51, 31, 19 and 13 pS. No clear difference was observed in the pattern of conductance levels displayed by neurones in which different subunit combinations were detected. 4. Whole‐cell agonist‐induced currents were strongly reduced by the NMDA‐R antagonist ifenprodil, at a concentration that mainly affects receptors containing NR2B in recombinant systems. Currents activated by NMDA had a high sensitivity to extracellular Mg2+. 5. The fraction of the total cation current through NMDA‐R that was carried by Ca2+, measured using a combination of patch clamp and fluorometry in neurones loaded with a high concentration of the Ca2+ indicator fura‐2, was found to be approximately 12%. 6. NMDA‐R‐mediated excitatory synaptic currents (EPSCs) had similar time courses to those in neurones in other brain regions. The decay kinetics were biexponential, with respective mean values for the fast (tau f) and slow (tau 8) time constants of 79 and 300 ms at ‐60 mV, and 66 and 284 ms at +40 mV. EPSCs were greatly reduced by ifenprodil (3 microM). 7. In conclusion, NMDA receptors in GABAergic medial septal neurones display a characteristic functional profile. The NR2 subunit mRNA detected and the single channel conductance levels observed suggest that, in addition to NR2B, which is present in nearly all cells, NR2A, NR2C and NR2D are also expressed. However, most of the functional properties of NMDA‐Rs in these neurones, including the strong inhibition by ifenprodil and Mg2+, the high fractional Ca2+ current, and the time course of the synaptic currents, are more consistent with those known for NR2B than for the other NR2 subunits. These results suggest that the NR2B subunit dominates over other NR2 subunits in determining the functional properties of NMDA‐Rs in these neurones.

DEPOLARIZATION-INDUCED CALCIUM SIGNALS IN THE SOMATA OF CEREBELLAR PURKINJE NEURONS

Cerebellar Purkinje neurons express voltage-gated Ca2+ channels that are located on their somata and dendrites. Previous reports, based on microelectrode recordings and fura-2 Ca2+ imaging, suggested that depolarization-mediated intracellular Ca2+ signaling is confined almost completely to the dendrites. We investigated the spatial distribution of depolarization-induced Ca2+ signals in Purkinje neurons by applying whole-cell patch-clamp recordings combined with fluorometric Ca2+ imaging to cerebellar slices. Under our recording conditions, depolarizing pulses produced the dendritic but also large somatic Ca2+ signals. By selective perfusion of the slice with a Ca(2+)-free EGTA-containing solution, we could isolate experimentally Ca2+ signals in somata and dendrites, respectively. Moreover, experiments performed on cerebellar slices from young rats (up to postnatal day 6), in which Purkinje neurons are almost completely devoid of dendrites, showed that Ca2+ currents produced by the activation of somatic Ca2+ channels are associated with Ca2+ transients similar to those seen in the somata of adult Purkinje neurons. Our results strongly indicate that the depolarization-induced somatic Ca2+ signals are caused by Ca2+ entry through voltage-gated channels located on the somatic membrane of Purkinje neurons.

Glutamate- and AMPA-mediated calcium influx through glutamate receptor channels in medial septal neurons

The Ca(2+)-fraction of the ion current flowing through glutamate receptor channels activated either by glutamate or by AMPA was determined in forebrain neurons of the rat medial septum. By combining whole-cell patch-clamp and fura-2 fluorometric measurements we found that, at negative membrane potentials and at an extracellular free Ca(2+)-concentration of 1.6 mM, the Ca(2+)-fraction of the current activated by glutamate is 5.7%. A pharmacological analysis of responses produced by ionophoretically-released glutamate demonstrated a large contribution of NMDA-receptors but a small contribution of AMPA/kainate receptors to these responses. Interestingly, also AMPA-mediated currents were associated with significant changes in Ca(2+)-sensitive fluorescence. The fractional Ca2+ current of AMPA-induced responses was 1.2 +/- 0.4% (n = 5).

Quantitative Calcium Imaging in Brain Slices

Studying tissue slices has obvious advantages for cellular physiology, as it avoids the need for an enzyme treatment used in many single cell studies and enables to study visually-identified cell types. Since the majority of cells in a brain tissue slice preserve their structure and their synaptic contacts, this preparation is particularly useful to elucidate the properties of the synaptic interaction. Using Ca2+ imaging techniques, many intriguing questions regarding, for example, the spatial localization and functional role of synaptically-mediated Ca2+ signals or functional properties of subcellularly localized receptor channels can be addressed. Furthermore, the technique can be combined with other methods, like whole-cell patch clamp recordings1–3 and single-cell RT-PCR,4,3 to obtain information about the cell type-specific properties of neural function in various regions of the brain.