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During postnatal development neurones display discharge behaviours that are not present in the adult, yet they are essential for the normal maturation of the nervous system. Neonatal CA1 pyramidal cells, like their adult counterparts, fire regularly, but excitatory GABAergic transmission drives them to generate spontaneous high-frequency bursts until postnatal day (P) 15. Using intracellular recordings in hippocampal slices from rats at P8 to P25, we show herein that as the network-driven burst activity fades out, most CA1 pyramidal cells become intrinsically bursting neurones. The incidence of intrinsic bursters begins to rise at P11 and attains a peak of 74% by P18–P19, after which it decreases over the course of a week, disappearing almost entirely at P25. Analysis of the effects of different voltage-gated Ca2+ and Na+ channel antagonists, applied focally to proximal and distal parts of developing neurones, revealed a complex burst mechanism. Intrinsic bursting in developing neurones results from ‘ping-pong’ interplay between a back-propagating spike that activates T/R- and L-type voltage-gated Ca2+ channels in the distal apical dendrites and persistent voltage-gated Na+ channels in the somatic region. Thus, developing pyramidal neurones transitionally express not only distinctive synaptic properties, but also unique intrinsic firing patterns, that may contribute to the ongoing formation and refinement of synaptic connections.
Although the capacity of CNS neurones to generate propagating action potentials appears already in utero (Spitzer & Ribera, 1998), their distinctive adult firing patterns are elaborated after birth (Spitzer et al. 2002). The postnatal maturation of intrinsic excitability accompanies and interacts with other developmental programmes that underlie neuronal growth, ramification of axonal and dendritic branches and formation and pruning of synaptic connections. During this period neurones display patterns of collective firing that are not ordinarily manifested by their mature counterparts. Yet, these immature patterns may be fine-tuned to carry out certain vital developmental functions (Moody, 1998). Identifying the transitional patterns of neuronal activity and their underlying intrinsic and synaptic mechanisms is therefore essential for understanding CNS development.
Adult hippocampal CA1 pyramidal cells normally fire a single spike when depolarized by a brief stimulus. In response to sustained stimuli they fire a train of spikes which adapts partially or completely (Schwartzkroin, 1975; Madison & Nicoll, 1984). Neonatal CA1 pyramidal cells fire in a similar mode, though they are less excitable than the mature neurones (Schwartzkroin & Kunkel, 1982; Costa et al. 1991). During the first 3–4 postnatal weeks their intrinsic excitability progressively increases. In this period action potentials become faster and larger. Likewise, the spike afterdepolarization (ADP) becomes progressively longer. However, it was noted that despite the monotonic changes in these measures of intrinsic excitability, many neurones temporarily manifest a tendency to burst fire during their maturation (Costa et al. 1991).
In this study we have investigated in detail developmental changes in the firing modes of CA1 pyramidal cells. We report that between P12 and P19, up to 74% of the neurones convert to a bursting mode after which (between P20 and P25) they rapidly revert to the adult pattern of regular firing. Moreover, we show that bursting in this transitional period results from ‘ping-pong’ interplay between a back-propagating spike that activates specific subtypes of voltage-gated Ca2+ channels in the apical dendrites and persistent Na+ channels in the somatic region. We discuss the possibility that this transitional mode of bursting may be important for the refinement of synaptic connections in developing hippocampal neurones.
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In this study we have identified a transitional period between P12 and P25 in which CA1 pyramidal cells first acquire, and than lose, the propensity to generate high-frequency bursts of two or more spikes as their minimal suprathreshold response. In the middle of this period, 74% of CA1 pyramidal cells display a bursting phenotype, compared to 3% before, and 6% after, this period. Given that bursting neurones have a unique impact on neuronal plasticity and integration (Kepecs & Lisman, 2003), such an increase in the fraction of bursters must have dramatic consequences for hippocampal development and maturation.
A tendency of developing CA1 pyramidal cells to fire spike doublets has been noted previously (Costa et al. 1991). Here we expand this initial observation and investigate its ionic mechanism. Our findings suggest that intrinsic bursting in developing neurones results from ‘ping-pong’ interplay between spike afterpotentials in soma and in distal apical dendrites, similar to that found in 4-AP-treated adult CA1 pyramidal cells (Magee & Carruth, 1999). According to this model, a spike initiated at the proximal axon back-propagates into the soma and apical dendrites (Spruston et al. 1995; Colbert & Johnston, 1996). The extent of dendritic invasion and depolarization by this spike depends on the density ratio of inward (Na+ and Ca2+) to outward (K+) currents that co-activate during this process (Johnston et al. 1999). In adult CA1 pyramidal cells this ratio is low, so that a back-propagating spike is attenuated in the proximal dendrite and its impact on the somatic spike ADP is small (Hoffman et al. 1997). In this case the ADP is derived predominantly from activation of proximal INaP and is too small to initiate a regenerative burst response (Azouz et al. 1996; Su et al. 2001). However, if the inward to outward current density ratio is high, as may be the case in developing neurones between P12 and P25, the back-propagating spike would be much less attenuated and may trigger a dendritic Ca2+ spike. The latter spike, in turn, spreads proximally and augments the INaP-driven spike ADP at the somatic region, leading to the generation of a spike burst.
Our results support this proposed scheme. Firstly, bursting is suppressed by TTX application to the apical dendrites, indicating a requirement for spike backpropagation. This effect of TTX cannot be due to inhibition of dendritic INaP, because riluzole, which blocks this current, has no effect on bursting when applied at the same location. Secondly, bursting is suppressed by application of Ni2+ or nimodipine to the distal apical dendrites, but not to the somatic region, demonstrating that dendritic Ca2+ electrogenesis is crucial for this process. Thirdly, bursting is suppressed by riluzole application to the somatic region, indicating that activation of INaP at or near the soma also is essential for the evolution of a spike burst.
In adult CA1 pyramidal cells, back-propagating spikes activate Ca2+ channels in the apical dendrites (Jaffe et al. 1992; Spruston et al. 1995), but do not ordinarily evoke regenerative Ca2+ spikes. Presumably such an escalation is prevented by co-activation of fast inactivating K+ channels (A-type), whose density along the apical dendrite increases with distance from the soma (Hoffman et al. 1997). Blocking these channels with millimolar 4-AP facilitates spike invasion into dendritic branches and allows the development of a dendritic Ca2+ spike that, in turn, triggers somatic bursting. The dendritic Ca2+ channels involved in this activity also are predominantly of the T/R- and L-type (Magee & Carruth, 1999). Here we show that natural bursting in developing neurones is generated by the same types of Ca2+ channels. These findings are consistent with the predominant expression of T/R-type Ca2+ channels in distal apical dendrites of CA1 pyramidal cells (Christie et al. 1995; Magee & Johnston, 1997; Kavalali et al. 1997). Though we could not differentiate pharmacologically between T- and R-type channels, it is more likely that T-type channels underlie bursting, given their much lower threshold of activation and slower deactivation kinetics (Randall & Tsien, 1997; Su et al. 2002). It is less clear why L-type Ca2+ channels in distal apical dendrites are critical for bursting, as these channels reportedly aggregate mostly in the soma and proximal dendrites (Hell et al. 1993; Magee & Johnston, 1995; Christie et al. 1995).
A high ratio of inward to outward current density in developing neurones may be due to over-expression of Ca2+ and Na+ channels and/or under-expression of K+ channels compared to adult values. In CA1 pyramidal cells, the density of Ca2+ channels increase after birth and reaches adult values at P13 and P20, respectively (Kortekaas & Wadman, 1997). These changes, as well as the presumed translocation of T-type Ca2+ channels from soma to apical dendrites (Thompson & Wong, 1991; Karst et al. 1993), may underlie the appearance of intrinsic bursting between P12 and P18. A developmental increase in the density of INaP may also contribute to the appearance of intrinsic bursting, as this current also appears essential for this behaviour. On the other hand, the disappearance of bursting between P18 and P25 may be due to a late increase in density of functional A-type K+ channels in apical dendrites, which would counteract the depolarizing actions of ICa. Indeed, blocking these channels with millimolar 4-AP induced Ca2+-dependent bursting in 18 of 20 adult CA1 pyramidal cells (C. Yue, S. Chen and Y. Yaari, unpublished observations). Our finding that BAY-K8644 induces Ca2+-dependent bursting that originates in apical dendrites in developing, but not in adult CA1 pyramidal cells, also suggests that the latter neurones are less prone to dendritic Ca2+ electrogenesis than their young counterparts.
In adult neocortical pyramidal cells, backpropagating spikes were shown to trigger a dendritic Ca2+ spike and somatic bursting, provided spike invasion of the apical dendrites coincided with a distal dendritic EPSP (Larkum et al. 1999). Presumably the depolarization of the apical dendrites either by a single backpropagating spike or by an EPSP, is insufficient to set off a local Ca2+ spike. However, when the two potentials coincide, the total depolarization of the dendrites is adequately large to elicit a local Ca2+ spike, which in turn triggers somatic bursting.
Neonatal CA1 pyramidal cells display spontaneous network bursting that is driven by depolarizing GABAergic postsynaptic potentials. This activity disappears by P15–P16 with the maturation of GABAergic inhibition (Ben-Ari, 2002). During network-driven bursts individual neurones generate a synaptically driven burst of high-frequency (up to 100 Hz) spikes associated with a large increase in intracellular Ca2+ concentration (Garaschuk et al. 1998). The marked increase in intrinsic bursting described in this report temporally parallels the disappearance of early network bursting and attains maximum expression a few days after network bursting fades out. It is interesting that this sequence of developmental events can be recapitulated in the adult brain by inducing intense network bursting (i.e. seizure activity) with the convulsant pilocarpine. A few days after such treatment, intrinsic bursting appears in over 50% of regular-firing CA1 pyramidal cells (Sanabria et al. 2001). Like the natural intrinsic bursts in developing neurones, intrinsic bursts induced by seizure activity in adult neurones also are Ni2+ sensitive (Su et al. 2002), and also require Ca2+ electrogenesis in the apical dendrites (C. Yue, H. Su and Y. Yaari, unpublished observations). The hypothesis that early network bursting triggers the molecular changes underlying the subsequent appearance of intrinsic bursting is based on these similarities.
It has been hypothesized that early network bursting is germane to the establishment of synaptic connectivity during postnatal development. Presumably the early population bursts furnish the necessary pre- and postsynaptic coincident activity for transforming silent synapses into active ones (Durand et al. 1996) and for strengthening weakly connected synapses (Kasyanov et al. 2004). It is therefore tempting to speculate that the appearance of intrinsic bursting in CA1 pyramidal cells serves a further important role in modifying synaptic connectivity. Indeed, the transitional period of intrinsic bursting is also a period of intense proliferation and ramification of apical dendrites and formation of excitatory synapses on dendritic spines in these neurones (Pokorny & Yamamoto, 1981a,b). Likewise, the ability of CA1 pyramidal cells to undergo long-term potentiation in response to high-frequency (100 Hz) stimulation of afferent fibres becomes consistent only after P12 (Harris & Teyler, 1984; Muller et al. 1989), which is about the time of first appearance of bursting pyramidal cells (Fig. 2B). What can be the role of intrinsic bursting in these processes? Many studies have shown that both activity-dependent synaptogenesis and long-term potentiation of synaptic strength require activation of postsynaptic N-methyl-d-aspartate (NMDA) receptors and an increase in intradendritic Ca2+ levels (Muller et al. 2002). Clearly, a postsynaptic spike burst driven by a dendritic Ca2+ spike would be extremely effective in unblocking of postsynaptic NMDA receptor channels by Mg2+ and in raising dendritic Ca2+ levels in comparison with a solitary back-propagating spike that only partially invades the apical dendrites (Magee & Johnston, 1997). Consequently, the transitional period of intrinsic bursting during postnatal development may also be a period of heightened NMDA receptor-dependent synaptic plasticity.