Synchronized network activity in developing rat hippocampus involves regional hyperpolarization-activated cyclic nucleotide-gated (HCN) channel function

Abstract

The principal form of synchronized network activity in neonatal hippocampus consists of low frequency 'giant depolarizing potentials' (GDPs). Whereas contribution of both GABA and glutamate to their generation has been demonstrated, full understanding of the mechanisms underlying these synchronized activity bursts remains incomplete. A contribution of the h-current, conducted by HCN channels, to GDPs has been a topic of substantial interest. Here we focus on HCN1, the prevalent HCN channel isoform in neonatal hippocampus, and demonstrate an HCN1 spatiotemporal expression pattern in both CA3 principal cells and interneurons that correlates with the developmental profile of GDPs. Abrogation of HCN physiological function in CA3, via the selective I(h)-blocker ZD7288, disrupts GDP generation. Furthermore, ZD7288 specifically abolishes spontaneous bursting of the CA3 pyramidal cells at frequencies typical of GDPs without major influence on interneuronal firing. These findings support a pivotal role for HCN channels expressed by CA3 neurons, and particularly CA3 pyramidal cells, in GDP-related network synchronization.

Fig. 1

Distinct developmental gradients of HCN1 channel expression exist in neonatal hippocampus. HCN1 expression was analysed in (A, D and G) CA3 and (C, F and I) dentate gyrus on (A–C) P3, (D–F) P6 and (G–I) P10. (A) In CA3, HCN1 protein was robustly expressed in pyramidal cells (sp) as early as P3. In addition, a large number of interneurons, mainly located in strata radiatum (sr) and oriens (so), expressed HCN1 in neonatal CA3 (arrows in A). (D and G) With maturation, HCN1 protein expression in pyramidal cells declined. Expression in interneurons remained robust (arrows in D and G) but became more prominent in interneurons of stratum pyramidale (arrowheads in G). (C) In dentate gyrus, few hilar interneurons expressed HCN1 on P3 (arrow). (F and I) Only in the second postnatal week did their number increase substantially (arrows). HCN1 expression was absent in granule cell layer (GCL). Quantitative analyses of HCN1-expressing interneurons (per CA3 or hilar area) on (B) P3, (E) P6 and (H) P10 demonstrated the earlier appearance of the channels in CA3 (blue bars) compared to the hilus (pink bars). These analyses further revealed developmental gradients of HCN1 expression along the longitudinal axis of the hippocampus. Septo-temporal differences were significant in CA3 on P3 (B, blue bars; one-way ANOVA, F_9,20 = 2.9, P = 0.03), and in hilus on P6 (E, pink bars; F_9,20 = 20.5, P < 0.001; y-axis, number of HCN1-positive cells/unit area; x-axis, position of sections according to inset in G). Inset in A, double-labelling of HCN1 (green) and GAD65 (red) revealed colocalization in virtually all (≈ 96%) neurons in CA3 stratum oriens on P3 (arrows), demonstrating their GABAergic nature. HCN1-expressing pyramidal cells (sp.) did not express GAD65. Inset in D, high magnification photograph of CA3 on P6, demonstrating HCN1-immunoreactivity in somata of interneurons (arrows) and in (presumably GABAergic) axon terminals innervating CA3 pyramidal cells perisomatically (arrowheads). Scale bar, 100 μm (A, C, D, F, G and I), 50 μ m (inset in A), 20 μ m (inset in D).

Fig. 2

Blockade of I_h channel function reduced GDP frequency. (A) Sample traces recorded from a CA3 pyramidal neuron, from a P4 rat, demonstrating the inhibitory effect of 1 μM ZD7288 on GDP frequency. The effect of ZD7288 persisted after washout of the compound (bottom trace). (B) Time course (1-min bin size) of the effect of 1 μM ZD7288 on GDP frequency. (C) Summary of the effect of increasing concentrations of ZD7288 on GDP frequency (n = 3–4).

Fig. 3

Effect of ZD7288 on neonatal CA3 pyramidal cells and interneurons. (A) Pyramidal cells were exposed to a 400-ms hyperpolarizing current step in the absence and presence of 1 μM ZD7288. Note the presence of the hyperpolarizing sag and rebound depolarization (RD) during and after the application of the hyperpolarizing pulse. Both the sag and rebound depolarization were blocked by a 7-min bath application of ZD7288. (B) Time course of the effect of 1 μM ZD7288 on voltage sag and rebound depolarization (n = 4). (C) ZD7288 (1 μM) altered spike bursting firing of CA3 pyramidal cells. (D) Time course of the effect of 1 μM ZD7288 on the number of spikes per burst (n = 4). (E) Even 40 μM ZD7288 did not alter the intrinsic firing frequency of CA3 interneurons, nor did it affect the frequency of GABA_A-mediated spontaneous and miniature postsynaptic currents (PSCs) arriving at CA3 pyramidal cells. (F) Summary of the effects of ZD7288 on average frequency of interneuronal spikes (n = 6) and GABA_A-mediated synaptic activity (spontaneous, n = 7; minis, n = 5).