Ih in CA1 and CA3 pyramidal neurones
In hippocampal pyramidal neurones, Ih is activated during voltage excursions negative to the resting potential. We examined Ih using whole-cell voltage clamp techniques, and, as illustrated in Fig. 1A, separated time-dependent Ih from time-independent (at this time scale) IK(ir) based on the differential sensitivity of the latter current to Ba2+. With IK(ir) blocked by bath application of 0.5 mm Ba2+, all time-dependent current recorded at voltages between −60 and −140 mV (centre) was sensitive to the antagonist ZD7288 (Gasparini & DiFrancesco, 1997) (not shown), and therefore identified as Ih.
Figure 1. Separation of Ih and IK(ir) A, two hyperpolarization-activated currents, Ih and IK(ir), were distinguished on the basis of differential sensitivity to low concentrations of Ba2+. On the left is total current recorded during 2.5-s-long steps from −40 mV to voltages between −50 and −110 mV (in 10 mV increments); in the centre is Ih, recorded in the presence of 0.5 mm Ba2+; on the right is Ba2+-sensitive IK(ir) determined by subtracting the previous two sets of traces. The Ba2+-resistant current was identified as Ih by its slow activation and deactivation kinetics, and its sensitivity to the blocker ZD7288 (100 μm) (not shown). These traces were not leak- or capacity-subtracted; the fast downward transients are synaptic currents not blocked in these recordings. B, current-voltage (I–V) relations for Ih () and IK(ir) () in CA1 and CA3 pyramidal neurones; measurements were taken at the end of 2.5-s-long hyperpolarizations from −40 mV. Ih amplitude was larger in CA1 pyramidal neurones (statistically significant at all voltages negative to −70 mV and almost so at −60 mV), its activation threshold was more positive (arrows), and its amplitude relative to IK(ir) was significantly greater (see Results). (n= 9 for CA1, 17 for CA3). All data in this figure were taken from slices cultured for 3–4 days from P0–P1, and are presented as mean ±s.d.; *P < 0.05, **P < 0.01, and ***P < 0.001.
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Ih in CA1 pyramidal neurones was significantly larger than in CA3 neurones at all voltages negative to and including −70 mV (Fig. 1B), and had a more positive activation threshold (near −50 mV versus near −60 mV in CA3 neurones; arrows). Further, Ih was proportionally larger than IK(ir) in CA1 neurones; the ratio of Ih to IK(ir) was 2.2 ± 1.0 in CA1 versus 1.0 ± 0.6 in CA3 at −110 mV (P < 0.001; all data are presented as mean ±s.d., n values indicated in legends where appropriate). Differences in this balance between Ih and IK(ir) may help differentiate excitability in CA1 and CA3 pyramidal neurones (see also Takigawa & Alzheimer, 2003).
Ih sensitivity to vitronectin
To investigate regulation of Ih, we either cultured P0–P1 hippocampal slices from Swiss-Webster mice in the presence or absence of added vitronectin (10 μg ml−1 to serum-free medium), or cultured slices from vitronectin-deficient mice (Zheng et al. 1995) of the same age and compared currents to those of the C57BL/6J background strain. In either case, we examined Ih and other currents after 3–4 days in culture. Figure 2A1 and 2 show representative traces from naive and vitronectin-exposed neurones CA1 and CA3 pyramidal neurones; Ih was recorded during hyperpolarizing steps to a test voltage of −100 mV. These records are superimposed to illustrate the larger amplitude and more rapid activation of Ih in vitronectin-exposed neurones.
Figure 2. Reciprocal sensitivity of Ih to vitronectin exposure and deficiency A1 and 2, representative traces recorded at −100 mV from naive and vitronectin-exposed Swiss-Webster CA1 and CA3 pyramidal neurones. Note that currents from vitronectin-exposed neurones are larger and activate more rapidly. As in Fig. 1, traces were not leak- or capacity-subtracted, and the downward transients are not-blocked synaptic currents. B1–3, I–V relations illustrating the larger Ih observed in vitronectin-exposed CA1 and CA3 neurones, and the reciprocally smaller Ih of vitronectin-deficient CA1 neurones. C1–3, plot percentage differences in Ih amplitude at each voltage for the I–V relations in panels B1–3, computed as ([(Ih(experimental)/Ih(naive/wild-type)) − 1]× 100). These are reasonably independent of voltage for vitronectin-exposed and vitronectin-deficient CA1 neurones, but monotonically increase with voltage in vitronectin-exposed CA3 neurones. The continuous line shows the differences predicted from the values of Gmax, V½ and k derived from Boltzmann analyses of G–V relations (Fig. 4A–C), the grey diamonds show the differences predicted from variations in Gmax only, and the grey lines show the differences predicted from variations in V½ and k only. See the text for further details. D1–3, times for currents to reach one-half steady-state amplitude (t½), and their reciprocal sensitivity to vitronectin exposure or deficiency. The inflection point for CA3 neurones (D3) roughly corresponds to the position of V½ (see Fig. 4C). nCA1:Swiss-Webster= 27 for naive, 25 for + vitronectin; nCA1:C57BL/6J= 14 for wild-type, 21 for vitronectin−/−; nCA3:Swiss-Webster= 21 for naive, 33 for + vitronectin. Data from these same cells were used for construction of Fig. 4 and Table 1. Data are mean ±s.d.; *P < 0.05, **P < 0.01, and ***P < 0.001.
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Ih amplitude in pyramidal neurones was sensitive to vitronectin: significantly larger in vitronectin-exposed neurones, and reciprocally, smaller in neurones from vitronectin-deficient slices (current-voltage, I–V, relations of Fig. 2B1–3). In CA1 neurones, percentage differences in mean Ih amplitude (open circles in Figs 2C1 and 2) were reasonably independent of voltage between −80 and −120 mV, showing increases of 20–38% with vitronectin exposure, and decreases of 20–25% for vitronectin deficiency. In CA3 neurones, increases in Ih amplitude showed a pronounced curvature with voltage, from 28% at to 120 mV to 104% at −80 mV (Figs 2 and 3).
Figure 3. Preferential vitronectin sensitivity of Ih Current amplitudes and densities in a population of CA1 and CA3 pyramidal neurones for which measures of Ih, IK(ir) and whole-cell capacitance (Cm) were all available. Currents were recorded during steps to −100 mV from −40 mV, and separated as described in Fig. 1. A, current amplitude; B, whole-cell capacitance Cm (as an index of somatic and proximal dendritic membrane area); C, current density expressed as pA/pF. Only Ih amplitude and density differed significantly between naive and vitronectin-exposed neurones. nCA1= 9 for naive, 9 for + vitronectin; nCA3= 17 for naive, 33 for + vitronectin. Parenthetically, vitronectin deficiency did not significantly affect Cm of CA1 neurones (61.2 ± 14.2 pF versus 60.7 ± 5.7 pF for wild-type; P > 0.05). Also, Cm of naive Swiss-Webster CA1 pyramidal neurones (47.8 ± 8.0 pF, n= 9) was significantly smaller than that of naive CA3 neurones (62.0 ± 15.8 pF, n= 17; P < 0.05) and that of CA1 neurones in slices from wild-type C57BL/6J mice (60.7 ± 5.7 pF, n= 15; P < 0.001). Data are mean ±s.d.; *P < 0.05, **P < 0.01, and ***P < 0.001.
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Ih activation was also sensitive to vitronectin: faster and slower with vitronectin exposure and deficiency, respectively (Fig. 2D1–3). For CA1 neurones, mean times to reach one-half the amplitude at the time of repolarization were 15–18% shorter between −80 and −120 mV in vitronectin-exposed neurones, with the maximum difference at −90 mV (P < at least 0.05 at all voltages), and 15–28% longer in neurones from vitronectin-deficient slices, with the maximum difference between −90 and −100 mV (P < at least 0.05 at all voltages). For CA3 neurones, one-half activation times were 9–30% shorter between −90 and −120 mV, with the maximum difference at −100 mV (P < at least 0.05 at all voltages).
Note that neurones derived from vitronectin-deficient mice were themselves responsive to vitronectin. For example, Ih amplitude at −100 mV in vitronectin-exposed vitronectin−/− CA1 neurones was 269.0 ± 40.2 pA versus 209.5 ± 31.9 pA for neurones in control vitronectin-deficient slices (P < 0.05; n= 5 for vitronectin−/−, 4 for +vitronectin), and in CA3 neurones was 73.6 ± 10.2 pA versus 54.8 ± 14.3 pA in control vitronectin−/− neurones (P < 0.05; n= 6 for vitronectin−/−, 7 for +vitronectin).
Exposure to vitronectin did not appear to affect somatic and proximal dendritic membrane area, as assessed by measurements of whole-cell capacitance (Cm). There were no statistically significant differences in Cm between naive and vitronectin-exposed CA1 neurones or CA3 neurones (Fig. 3B), nor between CA1 neurones in wild-type and vitronectin-deficient slices (Fig. 3 legend).
Further, the effects of vitronectin were confined to Ih; neither the amplitude or density of IK(ir) were altered by vitronectin exposure, which therefore shifted the balance between Ih and IK(ir) in favour of Ih. This is illustrated in Figs 3A and C, where Ih and IK(ir) amplitudes at −100 mV are compared. At this voltage, Ih amplitude will be sensitive to variation in both Gmax (maximum available conductance) and V½ (voltage of one half-activation as determined by fits to Boltzmann relations). In CA1 and CA3 neurones for which measures of IK(ir) along with Ih were available, mean Ih amplitude in vitronectin-exposed neurones was significantly larger, by 31.5% in CA1 and by 73.2% in CA3. Ih densities followed: 31.6% larger in CA1 and 70.8% larger in CA3. In these same neurones, there were no significant differences in IK(ir) amplitude or density.
Voltage dependence and kinetics of Ih activation
We examined the voltage-dependence of Ih activation by constructing conductance-voltage (G–V) relations using chord conductances calculated from steady-state current amplitudes (see Methods). Shown in Fig. 4A–C are G–V relations for CA1 and CA3 pyramidal neurones: naive or wild-type (open symbol), and vitronectin-exposed or -deficient (closed symbol). For these comparisons, conductance measurements for an individual cell were normalized relative to its Gmax. The continuous lines are fits of these values to Boltzmann relations; the positions of mean V½ (Table 1) are indicated by the vertical dotted lines. A and C show data for CA1 and CA3 neurones exposed to vitronectin (Swiss-Webster mice); B shows data for CA1 neurones in vitronectin-deficient slices (C57BL/6J mice).
Table 1. Boltzmann analyses of G–V relations with variation in vitronectin
In CA1 neurones (Figs 4A and B; Table 1), only Gmax was significantly sensitive to vitronectin, being 23.3% larger in vitronectin-exposed neurones and 17.9% smaller in vitronectin-deficient neurones. In these cells the positions of G–V relations (as indicated by V½) were not significantly altered by vitronectin exposure (displacement of +1.9 mV from −80.0 ± 4.9 mV to −78.1 ± 2.3 mV; P > 0.05) or vitronectin deficiency (displacement of −1.8 mV from −82.0 ± 2.9 mV to −83.8 ± 3.1 mV; P > 0.05). In CA3 neurones (Fig. 4C; Table 1), Gmax was 35.4% larger in vitronectin-exposed cells, and G–V relations showed a further statistically significant +3.0 mV displacement (from −92.8 ± 3.7 mV to −89.8 ± 5.8 mV; P < 0.05).
The behaviours of the G–V relations in Fig. 4A–C are reflected in the percentage changes in Ih amplitude shown in Fig. 2C1–3, where the continuous lines are calculated from differences in the I–V relations predicted from the three Boltzmann parameters Gmax, V½ and k. Clearly, in CA1 neurones, much of the difference in current amplitudes derives from differences in Gmax (shown as grey diamonds superimposed on the vertical axis), with variation in V½ and k (grey lines), contributing some increase only at more positive values. In CA3 neurones, the non-linear increases in Ih amplitude reflect differences in V½ and k in addition to the larger Gmax.
To examine kinetic differences illustrated in Fig. 2D1–3 in more detail, traces were fitted with the sum of two exponential functions: Ih=Afast exp (−t/τfast) +Aslow exp(−t/τslow), where Afast and Aslow are the amplitudes of two distinct kinetic components with time constants τfast and τslow. This function provided a good description of the time course of Ih activation, as illustrated in Fig. 5A, which shows pairs of representative traces (capacity- and leak-subtracted) from naive and vitronectin-exposed CA3 pyramidal neurones, scaled to match their maximum amplitudes, and with fitted functions superimposed in red.
Shown below are data summarizing the activation properties of Ih: the activation time constants τfast and τslow in Fig. 5B1–3, and the amplitudes Afast and Aslow in Fig. 5C1–3. The time constants τfast and τslow were voltage dependent, becoming more rapid at increasingly negative voltages, and differed by about an order of magnitude at all voltages.
Consider the data for naive CA1 and CA3 neurones, which indicate that two factors contribute to the slower activation of Ih in CA3 pyramids. First, the intrinsic time constants of CA3 neurones are slower. For example, in naive (black curves in Fig. 5B1 and 3) Swiss-Webster CA3 neurones at −90 mV, τfast was 552.5 ± 174.3 ms versus 323.1 ± 96.6 ms in CA1 (P < 0.05), and τslow was 5353.5 ± 1997.6 versus 2423.8 ± 513.2 in CA1 (P < 0.01). Second, the relative contribution of the fast component to total Ih is smaller in CA3 neurones, where Afast was often smaller than Aslow (compare and between Figs 5C1 and 3) and represented an average of 47.3 ± 8.4% (pooled across −90 to −120 mV) of total Ih. In CA1 neurones, Afast was always larger than Aslow and contributed an average of 63.1 ± 5.7% (pooled across the same voltages) to the total (P < 0.05 as compared to CA3).
Vitronectin, in contrast, did not appear to affect the values of time constants underlying Ih in either CA1 or CA3. As illustrated in Fig. 5B1–3 (compare black with red or green curves as appropriate), τfast and τslow did not differ between naive and vitronectin-exposed neurones, or between neurones derived from wild-type or vitronectin-deficient mice. Comparisons of mean values for τfast and τslow at each voltage did not reveal statistically significant differences (P > 0.05 at all voltages), nor did the slopes of τ–V relations for τfast and τslow (evaluated as mV per e-fold change) differ significantly (P > 0.05).
Rather, only the amplitudes of Afast and Aslow, and their relative contributions to total Ih, differed with vitronectin exposure or deficiency (Fig. 5C1–3). The average percentage difference in Afast (pooled values between −90 and −120 mV) in vitronectin-exposed neurones was +34.7 ± 10.4% in CA1 and +74.2 ± 44.9% in CA3, and in vitronectin-deficient neurones was −23.0 ± 5.2% in CA1 (P < 0.05 at virtually all voltages). Over these same voltages, Aslow was also consistently larger in vitronectin-exposed as compared to naive neurones (+18.7 ± 5.3% in CA1 and +7.8 ± 22.2% in CA3) and smaller in vitronectin-deficient as compared to wild-type neurones (−15.0 ± 9.8% in CA1), although these measurements were very variable and differences did not reach statistical significance. Nevertheless, at every voltage between −90 and −120 mV, percentage differences in Afast were larger than those of Aslow. Thus, embedded in differences in Ih amplitude seen with vitronectin exposure or deficiency is a consistent preferential sensitivity of Afast to vitronectin, and thus variation in its relative contribution to total Ih.
Immunochemical examination of HCN1 and HCN2
The potential molecular underpinnings of these variations in Ih include the four members of the HCN channel gene family expressed in mammalian neurones (HCN1, HCN2, HCN3 and HCN4; Santoro & Tibbs, 1999). HCN1 and HCN2 are highly expressed in hippocampal pyramidal neurones, and native hippocampal Ih channels are probably heteromultimers incorporating HCN1 and/or HCN2 in varying combinations with other subunits (see Discussion).
We therefore examined expression of HCN1 and HCN2 proteins in cultured naive and vitronectin-exposed slices by immunofluorescence. Shown in Fig. 6A are representative images of HCN1 immunoreactivity; grey-scale images are shown above and pseudocoloured images below. Slices were grown and processed in parallel, and images were acquired using identical parameters. Visual comparison suggests a generalized luminance increase throughout the vitronectin-exposed slice, with particular accumulations of HCN1 immunoreactivity in entorhinal cortex adjacent to the hippocampus (region a in Fig. 6A), in the striatum radiatum and striatum lacunosum-moleculare of CA1 (region b), and in an area of the slice adjacent to the fornix (region c).
Figure 6. Analyses of HCN1 and HCN2 immunoreactivity A, fluorescence images of HCN1 immunoreactivity in cultured hippocampal slices. Images are shown above using a greyscale, and below using a pseudocolour scale to better illustrate luminance differences between the naive and vitronectin-exposed slices. B and C, cumulative probability histograms of HCN1 and HCN2 immunoreactivity, quantified as pixel luminance normalized to the average pixel luminance of the naive slices in each set of cultures (see Methods). Shown in each case are background luminance (black; determined by antigen absorption of the primary antibody or use of the secondary antibody only), and luminance of naive (blue) and vitronectin-exposed (red) slices. The vertical broken lines indicate median values of relative luminance; the naive values are not exactly 1.00 because these are median values of luminance normalized to appropriate means. The HCN1 luminance histogram is clearly shifted towards brighter values in vitronectin-exposed slices, while the HCN2 luminance histograms essentially superimpose. nHCN1= 8 for naive, 7 for + vitronectin, nHNC2= 8 for naive, 8 for + vitronectin; each analysed in two independent sets of cultures. See Results for further details.
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Semi-quantitative comparisons of immunoreactivity in naive and vitronectin-exposed slices suggest a selective increase in HCN1 immunoreactivity, with little change seen in HCN2 levels. In each experiment, we measured the luminance of each pixel in groups of naive and vitronectin-exposed slices, and normalized these values against the mean luminance of the naive slices (see Methods). Viewed as cumulative probability histograms of relative pixel luminance (Figs 6B and C), the rightward shift in the histogram for HCN1 immunoreactivity suggests higher HCN1 levels in vitronectin-exposed slices. In contrast, the histograms for HCN2 immunoreactivity virtually superimpose, suggesting little difference in HCN2 levels. Increases in mean HCN1 luminance were consistent with this interpretation, but were trends that did not reach statistical significance: relative HCN1 pixel luminance increased ∼15% from 1.00 ± 0.22–1.15 ± 0.21, while for HCN2, relative pixel luminance showed virtually no change (from 1.00 ± 0.18–1.03 ± 0.14). Median relative pixel luminance values showed a similar pattern (vertical lines to baseline in Figs 6B and C): increase of ∼15% from 0.96 ± 0.23–1.11 ± 0.21 for HCN1 and little or no change between 1.02 ± 0.19 and 1.04 ± 0.15 for HCN2.