CB1 receptor-dependent and -independent inhibition of excitatory postsynaptic currents in the hippocampus by WIN 55,212-2

We investigated the effect of a synthetic cannabinoid, WIN 55,212-2 on excitatory postsynaptic currents (EPSCs) evoked by stimulation of Schaffer collaterals in CA1 pyramidal cells. Bath application of WIN 55,212-2 reduced the amplitude of EPSCs in dose-dependent manner tested between 0.01 nM and 30 μM. In rats and mice, this cannabinoid ligand inhibited excitatory synapses in two steps at the nM and μM concentrations. When the function of CB1 cannabinoid receptors (CB1R) was impaired, either by the application of a CB1R antagonist AM251, or by using CB1R knockout mice, WIN 55,212-2 in μM concentrations could still significantly reduced the amplitude of EPSCs. WIN 55,212-2 likely affected the efficacy of excitatory transmission only at presynaptic sites, since both at low and high doses the paired pulse ratio of EPSC amplitude was significantly increased. The inactive enantiomer, WIN 55,212-3, mimicked the effect of WIN 55,212-2 applied in high doses. In further experiments we found that the CB1R-independent effect of 10 μM WIN 55,212-2 at glutamatergic synapses was fully abolished, when slices were pre-treated with ω-conotoxin GVIA, but not with ω-agatoxin IVA.
1
Introduction
The type 1 cannabinoid receptors (CB1Rs) have been shown to control the release of different neurotransmitters, but the mechanisms underlying the regulation of synaptic communication could substantially vary between brain regions (Freund et al., 2003). Pharmacological results, suggesting a presynaptic locus of action of cannabinoid receptor ligands, have been fully supported by immunohistochemical data. Several studies demonstrated at the electron microscopic level that CB1Rs decorated both inhibitory and excitatory axon terminals (Katona et al., 1999, 2006; Kawamura et al., 2006). In addition, recent high-resolution quantitative studies established that CB1Rs were found all around the axon membrane, but were enriched in the perisynaptic annulus and on preterminal segments, whereas immunolabelling was weaker in the synaptic active zone (Nyiri et al., 2005; Kawamura et al., 2006). This subcellular distribution of CB1Rs might imply an action on several regulatory mechanisms of transmitter release, including the control of Ca2+ entry via voltage-dependent Ca2+ channels (primarily by receptors located in the perisynaptic annulus), the reduction of axonal conduction (by receptors present on the preterminal segments), or a direct action on exocytosis (Wilson et al., 2001; Diana and Marty, 2003).
In spite of the direct anatomical evidence, several pharmacological observations suggest that some synthetic cannabinoid agonists (mainly WIN 55,212-2) could also have a CB1R-independent action on synaptic glutamate release. This possibility has been fuelled primarily by experiments using CB1R knockout mice. Our laboratory was the first to show that, in the absence of CB1Rs, WIN 55,212-2 was still able to reduce excitatory, but not inhibitory postsynaptic currents in CA1 pyramidal neurons (Hájos et al., 2001). Moreover, WIN 55,212-2 was more potent in suppressing GABAergic than glutamatergic transmission (Hoffman and Lupica, 2000; Ohno-Shosaku et al., 2002; Hájos and Freund, 2002), providing further support for the possible presence of CB1R-independent binding site at excitatory synapses. Importantly, AM251, a CB1R antagonist prevented the reduction of synaptic inhibition after application of WIN 55,212-2, whereas glutamatergic transmission could still be suppressed by about 50% in the presence of AM251 (Hájos and Freund, 2002). In contrast to the above findings showing that hippocampal glutamatergic synapses were effectively regulated independent of CB1Rs, electrophysiological data from other groups suggested that CB1Rs were solely responsible for the cannabinoid modulation of excitatory synaptic transmission in the hippocampus (Ohno-Shosaku et al., 2002; Domenici et al., 2006; Takahashi and Castillo, 2006).
To shed light on the reasons behind the contradictory findings regarding the involvement of CB1R-dependent vs. -independent mechanisms in the regulation of hippocampal excitatory synapses, we re-examined the effect of WIN 55,212-2 on monosynaptically evoked excitatory postsynaptic currents (EPSCs) in CA1 pyramidal cells. All these experiments were performed in a modified submerged recording conditions (Hájos et al., 2005).
2
Methods
Experiments were carried out according to the guidelines of the institutional ethical code and the Hungarian Act of Animal Care and Experimentation (1998. XXVIII. section 243/1998.). Male Wistar rats (14–18 days old), as well as wild type and CB1R knockout mice (15–25 days old, CD1 strain) were used. The animals were deeply anaesthetized with isoflurane followed by decapitation. After opening the skull, the brain was quickly removed and immersed into ice-cold cutting solution containing (in mM: sucrose 252; KCl 2.5; NaHCO3 26; CaCl2 0.5; MgCl2 5; NaH2PO4 1.25; glucose 10). The solution had been bubbled with 95% O2/5% CO2 (carbogen gas) for at least 30 min before use. Thick horizontal slices (350 μm from mice and 400 μm from rats) were prepared using a Leica VT1000S Vibratome. The CA3 region was removed to prevent epileptic burst firings. The slices were stored in an interface type chamber containing ACSF (in mM: 126 NaCl, 2.5 KCl, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, and 10 glucose) at room temperature for at least 1 h before recording. After the initial incubation period, slices were transferred individually into a submerged type recording chamber.
Whole-cell patch-clamp recordings were obtained at 30–32 °C from CA1 pyramidal cells visualized by infrared DIC videomicroscopy (Zeiss Axioscope, Germany). Patch electrodes were pulled from borosilicate glass capillaries with an inner filament (1.5 mm O.D.; 1.12 mm I.D., Hilgenberg, Germany) using a Sutter P-87 puller. Electrodes (∼3–6 MΩ) were filled with a solution containing (in mM) 80 CsCl, 60 Cs-gluconate, 3 NaCl, 1 MgCl2, 10 HEPES, 2 Mg-ATP, and 5 QX-314 (pH 7.2–7.3 adjusted with CsOH; osmolarity 275–290 mOsm). Excitatory postsynaptic currents (EPSCs) were recorded at a holding potential of −65 mV. Slices were perfused with ACSF containing 70–100 μM picrotoxin to block inhibitory neurotransmission. The solution was bubbled with carbogen gas at room temperature and perfused at a flow rate of 3–4.5 ml/min in a slice chamber optimized for laminar flow to ensure the stability of the amplitude of evoked currents and a better oxygenation of submerged slices (Hájos et al., 2005). To evoke EPSCs, the stimulating electrode was placed in the stratum radiatum of CA1. Pairs of electrical stimuli separated by 50 ms were delivered via a theta glass pipette (Sutter Instrument Company, Novato, CA) filled with ACSF at 0.1 Hz using a Supertech timer and isolator (Supertech LTD, Pécs, Hungary, http://www.superte.ch). Access resistances (between 4 and 18 MΩ, compensated 65–70%) were frequently monitored and remained constant (±20%) during the period of analysis. Signals were recorded with a Multiclamp 700A (Molecular Devices, Sunnyvale, CA), filtered at 2 kHz, digitized at 6 kHz (National Instruments PCI-6024E A/D board, Austin, TX), and analyzed off-line with the EVAN program (courtesy of Prof. I. Mody, UCLA, CA).
The drug was perfused in a given concentration until the maximal effect was reached. The time needed for maximal inhibition was usually 6–8 min. To avoid the possible effect of a changing pH, we added the same amount of HCl to the control solution. The concentration response relationship for WIN 55,212-2 was obtained as follows: control EPSC amplitudes in a 2–3 min time window were compared to those measured after 10 min drug application for the same period of time. Only those experiments were included that had stable amplitudes at least for 10 min before drug application. After each experiment, the tubing made of Teflon was washed with ethanol for 10 min and with ACSF for 15 min. Each data point represents the mean ± SEM of the maximal inhibition of the evoked EPSCs (n = 3–7). EC50 values were estimated by fitting a curve to the points of the dose response plots obtained in rats or wild type mice using the equation of f(x) = a/(1 + exp(−(x − c)/b)) + (100 − a)/(1 + exp(−(x − e)/d)), where ‘c’ and ‘e’ give the values for high and low affinity binding sites, respectively. The data points obtained in the presence of AM251 or in CB1 knockout mice were fitted by the equation of f(x) = a/(1 + exp(−(x − c)/b)), where ‘c’ gives the value of EC50. The curve fitting was done using Origin 7.5 (OriginLab Corporation, MA). The paired pulse ratio was calculated from the mean amplitude of the second EPSCs divided by the mean amplitude of the first EPSCs. The paired pulse ratio after drug treatment was compared with the control using Wilcoxon matched pairs test in Statistica 6.1 (Statsoft, Inc., Tulsa, OK). Data are presented as mean ± SEM.
Picrotoxin, WIN 55,212-2 and WIN 55,212-3 were purchased from Sigma-Aldrich, AM251 was obtained from Tocris, while ω-conotoxin GVIA and ω-agatonix IVA from Alomone Labs. For all experiments, WIN-55,212-2 was dissolved in 0.1N HCl giving a 20 mM stock solution stored at 4 °C. AM251 was dissolved in DMSO (100 mM) and stored at −20 °C. WIN 55,212-3 dissolved in DMSO (100 mM) was stored at 4 °C. From these stock solutions, the final dilution of drugs was done in ACSF containing picrotoxin under constant stirring and the prepared solution was bath applied. In control solutions, the vehicle was diluted in the same concentration as in the solutions containing drugs. Bovine serum albumin (BSA) was added in a concentration of 0.1 mg/ml to the solutions used for experiments with WIN 55,212-3.
3
Results
The effects of the cannabinoid agonist WIN 55,212-2 on EPSCs evoked by focal stimulation of Schaffer collaterals were measured in hippocampal CA1 pyramidal cells. First we performed concentration response analyses for the inhibitory effects of WIN 55,212-2 on evoked EPSC in rat slices (Fig. 1a). WIN 55,212-2 bath applied between the concentrations of 0.1 nM and 30 μM suppressed the amplitude of EPSCs in two steps. The apparent EC50 values from the fitted curve were 2.91 nM and 3.77 μM (Fig. 1c). Then we investigated the WIN 55,212-2-sensitivity of EPSCs, when AM251, a CB1R specific antagonist was added to the bath solution in the concentration of 2 μM. In spite of the presence of AM251, the cannabinoid agonist could still reduce the amplitude of evoked currents, but only in the μM range (Fig. 1a). The estimated EC50 value for this effect was 1.69 μM (Fig. 1c).
In the next set of experiments, we examined the concentration response relationship for the WIN 55,212-2-induced reduction of evoked EPSCs in mouse slices (Fig. 1b). The sensitivity of synaptic currents for WIN 55,212-2 was tested between the concentrations of 0.01 nM and 30 μM. Similar to that observed in rat slices, the cannabinoid agonist also decreased the amplitude of EPSCs in two steps. The EC50 values estimated by fitting a curve to the points of the dose response plot were 1.91 nM and 12.1 μM (Fig. 1c). To reveal whether WIN 55,212-2 could still suppress excitatory transmission in CB1R-independent manner in mice, we examined the effect of the cannabinoid agonist in CB1R knockout animals. As shown in Fig. 1b, WIN 55,212-2 effectively reduced the amplitude of EPSCs, but only in the μM range. The apparent EC50 value estimated from the curve fitting was 8.32 μM (Fig. 1c).
These results obtained both in rats and mice suggest that WIN 55,212-2 in nM concentrations inhibits excitatory synaptic transmission exclusively via CB1Rs, whereas in μM concentrations it has a mixed CB1R-dependent and -independent effect on glutamatergic transmission at Schaffer collateral synapses.
By a comparison of the paired-pulse ratios of evoked EPSCs, we next investigated whether the CB1R-independent action of WIN 55,212-2 is presynaptic, i.e. whether it is inhibiting glutamate release similar to that seen earlier for CB1Rs. We first examined the effect of 10 nM WIN 55,212-2 on the paired-pulse ratio in rats and wild type mice. After drug application, the ratio significantly increased to 132.5 ± 9.4% of control in rats and to 129.5 ± 14.2% of control and mice (Fig. 2; n = 5 each, p < 0.05, Wilcoxon test). These data are in line with both electrophysiological and anatomical results, suggesting a presynaptic locus of CB1R-dependent action. To check that the changes in the paired-pulse ratio were due to the activation of CB1Rs, we contrasted these values before and after the application of 10 nM WIN 55,212-2 in the presence of AM251. As expected, the paired-pulse ratio was not altered (98.4 ± 6.1%, n = 6; p > 0.1, Wilcoxon test; Figs. 2a,c). Next we compared the paired-pulse ratio before and after the application of 30 μM WIN 55,212-2. The ratio of evoked currents was significantly increased to 145.1 ± 5.3% of control in rat slices (n = 5; p < 0.05, Wilcoxon test) and to 141.3 ± 11.8% in slices from wild type mice (n = 7; p < 0.05, Wilcoxon test). To reveal that the CB1R-independent action of WIN 55,212-2 also modifies transmitter release, we investigated the paired-pulse ratio after application of 30 μM WIN 55,212-2, while 2 μM AM251 was included in the bath. The ratio of the amplitude of evoked EPSCs still significantly increased, to 136.4 ± 8.4% of control (Figs. 2a,c; n = 7, p < 0.05, Wilcoxon test). Similar to these results, 30 μM WIN 55,212-2 also raised the paired pulse ratio to 121.1 ± 2.9% of control in CB1R knockout mice (Figs. 2b,c; n = 6, p < 0.05, Wilcoxon test). Thus, the CB1R-independent effect of WIN 55,212-2 also appears to be presynaptic, reducing glutamate release from Schaffer collateral terminals.
As reported earlier (Shen and Thayer, 1998), WIN 55,212-2 in μM concentrations could directly alter Ca2+ currents independent of CB1Rs, an effect that could be mimicked by its inactive enantiomer WIN 55,212-3. To test whether at glutamatergic axon terminals a similar mechanism would be responsible for the reduction of EPSC amplitude, WIN 55,212-3 was bath applied in two different concentrations to rat slices. This inactive enantiomer significantly suppressed the amplitude of evoked EPSCs by 42.8 ± 10.7% (n = 5) and 54.7 ± 12.1% (n = 3) in 10 μM and 30 μM concentrations, respectively (Fig. 1c). These effects were indistinguishable from those values, which were obtained in the presence of AM251 after application of 10 μM (46.9 ± 7.8%; n = 5) or 30 μM (59.6 ± 3.2%; n = 4) WIN 55,212-2 (p > 0.1, Mann–Whitney U-test). These results suggest that the CB1R-independent action of WIN 55,212-2 on glutamatergic transmission might be due to the direct inhibition of Ca2+ entry into the presynaptic boutons.
To get deeper insight into the mechanisms underlying the CB1R-independent effects of WIN 55,212-2, we specifically examined the involvement of voltage-gated Ca2+ channels in this process. Rat slices were pre-incubated either in 250 nM ω-agatoxin IVA (a specific blocker of P/Q-type Ca2+ channels) or in 250 nM ω-conotoxin GVIA (a specific inhibitor of N-type Ca2+ channels) at least for an hour. After placing the pre-treated slices in the recording chamber, we bath applied 10 μM WIN 55,212-2 in the presence of 2 μM AM251. In slices pre-treated with ω-agatoxin IVA, the amplitude of EPSCs was reduced by 39.7 ± 8.3% (n = 4, p < 0.05, Wilcoxon test; Fig. 3a), whereas there was no change in the synaptic currents after application of WIN 55,212-2 in slices pre-incubated with ω-conotoxin GVIA (95.8 ± 3.2% of control, n = 6, p > 0.1, Wilcoxon test; Fig. 3b). The results of these experiments suggest that the CB1R-independent action of WIN 55,212-2 at glutamatergic synapses is mediated via inhibition of N-type Ca2+ channels.
4
Discussion
Our data presented here demonstrate that excitatory synapses of Schaffer collaterals in CA1 pyramidal cells are inhibited by WIN 55,212-2 both via CB1R-dependent and independent mechanisms. In low nM concentrations, this cannabinoid ligand solely acts as a CB1R agonist reducing glutamate release. In contrast, WIN 55,212-2 in the μM range suppresses glutamatergic synaptic transmission via activation of CB1Rs as well as inhibiting N-type Ca2+ channels independent of CB1Rs.
Shen et al., (1996) were the first to show that excitatory transmission in the hippocampus could be reduced by WIN 55,212-2, a finding that has been strengthened later by several other laboratories (Misner and Sullivan, 1999; Al-Hayani and Davies, 2000; Hájos and Freund, 2002; Ohno-Shosaku et al., 2002; Hoffman et al., 2003). Similarly to that observed in the hippocampus, WIN 55,212-2 was also shown to suppress excitatory synapses in other brain regions, including the cerebellum (Levenes et al., 1998; Takahashi and Linden, 2000), neocortex (Domenici et al., 2006), basolateral amygdala (Azad et al., 2003; Domenici et al., 2006), or striatum (Gerdeman and Lovinger, 2001; Huang et al., 2001). In earlier studies, the lack of immunostaining for CB1Rs at excitatory terminals (Katona et al., 1999; Hájos et al., 2000), taken together with experiments showing that WIN 55,212-2 could significantly reduce glutamate release in CB1R knockouts (Hájos et al., 2001; Kofalvi et al., 2003), fuelled the concept that distinct cannabinoid receptors control synaptic excitation and inhibition. This interpretation was supported by the unequivocal demonstration of high densities of CB1Rs on GABAergic axons, while adjacent glutamatergic terminals remained negative (Katona et al., 1999; Hájos et al., 2000; Nyiri et al., 2005), as well as by the complete disappearance of cannabinoid sensitivity of IPSCs in CB1R knockouts (Hájos et al., 2000, 2001; Varma et al., 2001; Wilson et al., 2001). Moreover, Hájos and Freund (2002) showed that AM251 could fully antagonise the effect of WIN 55,212-2 at GABAergic, but not at glutamatergic synapses, further strengthening the existence of a novel cannabinoid-sensitive binding site at hippocampal excitatory synapses. Recently this concept was substantially challenged both by anatomical and electrophysiological experiments, including studies from our own laboratory. First, using a different type of antibody, CB1Rs were convincingly shown to be present on glutamatergic terminals, although in much smaller quantities than on GABAergic axons (Katona et al., 2006; Kawamura et al., 2006). Specificity of the staining has been confirmed in CB1R knockout tissue. Second, excitatory transmission was found to be insensitive to the application of WIN 55,212-2 in distinct strains of transgenic mice lacking CB1Rs (Ohno-Shosaku et al., 2002; Domenici et al., 2006; Takahashi and Castillo, 2006). The discrepancy between earlier and recent data might be resolved by the present findings suggesting that at low nM concentrations WIN 55,212-2 specifically activates CB1Rs, whereas in the μM range the agonist could further reduce glutamate release via direct inhibition of presynaptic Ca2+ entry independent of CB1Rs (present study; Shen and Thayer, 1998; Kofalvi et al., 2007). This difference in the specificity of WIN 55,212-2 as a function of its concentration can be noticed already in studies reported by the Kano laboratory. WIN 55,212-2 in 100 nM caused a large reduction in the amplitude of EPSCs in wild type mice, but in CB1R knockouts less then 5% suppression was found (Ohno-Shosaku et al., 2002). However, in 2 μM concentration, WIN 55,212-2 inhibited excitatory transmission by about 20% in CB1R knockout mice, an effect that was unaltered in the presence of CB1R antagonists (Kawamura et al., 2006). Further support to the hypothesis that WIN 55,212-2 in μM concentrations can reduce excitatory transmission via CB1R-independent mechanisms come from the work of Hoffman et al. (2005), where the authors showed that in the presence of AM251, 3 μM WIN 55,212-2 significantly (appr. by 40%) reduced excitation. This view, however, is not supported by two recent studies using CB1R knockout animals, in which 5 μM WIN 55,212-2 was found to be completely ineffective at excitatory synapses (Domenici et al., 2006; Takahashi and Castillo, 2006). The explanation for these negative findings remains to be investigated.
Another finding of the present study that deserves discussion is that the effective concentration of WIN 55,212-2 that significantly inhibited the amplitude of synaptic events in slice preparations was 100 fold lower than it was earlier reported by several laboratories using similar recording circumstances (Takahashi and Linden, 2000; Robbe et al., 2001; Hájos and Freund, 2002; Hoffman et al., 2005). Compared to earlier studies, we changed some conditions that could account for the distinct efficacy of WIN 55,212-2, which allowed clearly separating CB1R-dependent and independent effects. The preparation and storage of slices, as well as the flow rate of the solution during recordings was modified: slices were cut in a sucrose containing solution and stored in an interface-type chamber before recordings, and a higher flow rate was used in the recording chamber, ensuring a better oxygenation of the tissue (Hájos et al., 2005). Under these circumstances, the amplitude of evoked synaptic currents became more stable, and, more importantly, the efficacy of WIN 55,212-2 to suppress excitatory synapses was comparable with those measured in cell cultures (Ohno-Shosaku et al., 2002) or binding assays (Felder et al., 1995).
Several studies in different brain regions suggested that the CB1R-independent effect of WIN 55,212-2 might significantly alter synaptic communication among neurons (Hájos et al., 2001; Pistis et al., 2004; Kofalvi et al., 2005; Matyas et al., 2006), presumably through a direct blockade of Ca2+ entry at the presynaptic terminals (Shen and Thayer, 1998; Kofalvi et al., 2007). In the present study, we provided evidence that the CB1R-independent effect of WIN 55,212-2 at glutamatergic synapses was mediated by inhibiting N-type Ca2+ channels.
The question arises whether WIN 55,212-2 in high doses could also alter GABAergic transmission independent of CB1Rs, since GABA release from CB1R-expressing axon terminals is known to depend on N-type Ca2+ channel activation (Wilson et al., 2001). Indeed, we found that in CB1R knockout mice 10 μM WIN 55,212-2 substantially reduced the amplitude of IPSCs to 62.8 ± 20.5% of control (n = 4). In contrast, when CB1R function was intact, both low and high doses of WIN 55,212-2 led to a comparable reduction of IPSC amplitudes (in 3 nM: 47.9 ± 15.4% of control, n = 4; in 10 μM: 45.2 ± 19.7% of control, n = 4), similar to results obtained earlier (Hájos and Freund, 2002). The reason why WIN 55,212-2 in 10 μM concentration did not result in an additional suppression of inhibitory events in the presence of functional CB1Rs may be explained by the fact that CB1R-dependent inhibition of GABAergic currents is entirely due to blocking N-type Ca2+ channels, which occludes the CB1R -independent action of WIN 55,212-2 directly on the same Ca2+ channels. Testing this hypothesis, and other novel aspects of cannabinoid modulation of GABAergic transmission is the subject of another line of investigations in our laboratory.
The question arises whether at glutamatergic synapses, under some experimental conditions, endocannabinoids can reduce the efficacy of neurotransmission via CB1R-dependent and -independent mechanisms, similar to high concentrations of WIN 55,212-2, which can modify Ca2+ entry directly. Some results indeed imply that endocannabinoids could directly inhibit different types of voltage-gated Ca2+ channels independent of CB1Rs (Chemin et al., 2001; Fisyunov et al., 2006), yet other data suggest that endocannabinoids released upon depolarization of a postsynaptic neuron (or exogenously applied) are unable to suppress excitatory transmission in CB1R knockout mice (Ohno-Shosaku et al., 2002; Straiker and Mackie, 2005, but see Rouach and Nicoll, 2003). Nevertheless, the importance of identifying a CB1R-independent binding site for WIN 55,212-2 as N-type Ca2+ channels at excitatory terminals lies in resolving some contradictions in pharmacological and behavioural studies that emerged partly due to the use of WIN 55,212-2 in widely varying concentrations (see a thorough discussion of this issue in Haller et al., 2007).

hippocampus
cannabinoids
pyramidal cell
glutamate
transmitter release
brain slices


Definition of the “Health Benefit Basket” in Poland

The subject of “health benefit basket” has been hotly debated for years among the Polish public, but until recently the debate has tended to be largely theoretical and abstract and therefore has lacked an effect on public policy. The situation changed in 2004, for two reasons: first the verdict of the Constitutional Tribunal invalidating the existing health insurance law and, second, Poland’s accession to the European Union. The first problem was solved in part by defining a list of specific exclusions in the law and a promise to establish an institution for health technology assessment. The second issue remains open, although to some extend it is being dealt with legally by regulations issued from the Ministry of Health on acceptable waiting times for health services.
Fundamentals of the health care system
Health care in Poland remains the responsibility of various sectors, but it is carried out principally by the social security system. Most aspects of the general health care framework were founded in 1997 by Article 68 of the Constitution which states that: (a) Everyone has a right to health care. (b) Citizens, regardless of their economic status, shall be provided with equal access to health care services financed from the public funds by the public authorities; the conditions and the scope of services are to be detailed in the appropriate laws. (c) Public authorities are obliged to provide special health care services to children, pregnant women, disabled persons, and the elderly. (d) Public authorities are obliged to counter epidemic diseases and prevent potentially health-threatening outcomes of environmental degradation.
Prior to 1989 the Polish health care system fell under a public integrated model, sometimes called the “Semashko” model, named for its Soviet ideologist, and changed gradually over time. In this system the government was both the principle insurer and the major provider of services, via health facilities owned and administered by regional representatives of the government (Voivods, “Governors”). The benefit basket in a given area was determined by the physical presence or absence of specialists and equipment. The country had begun to reform the health care system as early as the 1980s, albeit rather slowly. Until the end of the 1990s the reform process focused basically on redefining the role of the state and introducing quasimarket mechanisms, greater individual freedom, and decentralized responsibility and management. On 1 January 1999 the institutions of universal health insurance commenced their activity by virtue of the Law of Universal Health Insurance of 1997, with amendments from 1998. This established 16 Regional Health Insurance (Sickness) Funds along with the Health Insurance Fund for so-called “uniformed workers” (army workers, policemen, railway workers) and their families, which operated at interregional level. According to the Law of Universal Health Insurance, Health Funds sign contracts with health care institutions. Regional Health Funds covered particular voivodships (administrative regions) populated by 1–6 million inhabitants. After 2000 there were no legal barriers to the Health Funds extending their activities to other regions. Citizens were free to choose the Health Fund irrespective of their place of living. Health Funds had become autonomous organizational and property institutions, but they still remained public and managed funds collected through premiums under the supervision of a public “boards,” with the principle aim of providing insofar as their budget allowed the best health care to all insured persons in their region.
In reality, however, there was little competition between Health Funds for patients, except in some neighboring regions when certain Health Funds began absorbing persons in adjacent areas. Still however, they were focused rather on preserving quality and “proper relations” with providers in their own regions. In 2001 the Social-Democratic Government initiated a centralization of the system by merging all Health Funds into a unified universal National Health Fund (NHF) with 16 regional branches. The process of recentralization of the system lasted 2 years and was burdened with substantial technical problems and shortcomings. During the period from 2001 until 2005 there were six Ministers of Health, and the Health Insurance Act was completely re-written twice, one after the intervention of the Constitutional Tribunal. One of the reasons given by the Tribunal in declaring the law unconstitutional was the lack of definition concerning “conditions and scope of services” to which an insured person is entitled under the health insurance law.
Regulation of the benefit basket
There are various legal acts and documents defining the scope of services and conditions required for entitlement. In the hierarchy of regulations the most important and most general are those based upon the Constitution. More specific are the laws ratified by Parliament and the President, which regulate certain areas of social life and activities. The laws listed below include obligations of public organs and institutions, to provide and ensure health benefits to persons in need:Health insurance law which regulates the universal and obligatory insurance scheme that covers some 99% of Polish citizens and a large proportion of noncitizens, residents, both short- and long-term (Ustawa, 27 August 2004, r. o świadczeniach opieki zdrowotnej finansowanych ze środków publicznych, Dz.U. Nr 210 z 2004 r. poz. 2135).Labor code and occupational medicine law which regulates relations between employers and employees, including the health protection measures that employers are obliged to pay (Ustawa, 26 June 1974, r. Kodeks pracy-tekst jednolity, Dz.U. nr 21 z 1998 r. poz.94 z późn.zm; (Ustawa, 27 June 1997, o służbie medycyny pracy r., Dz.U. Nr 96 poz.593).Laws which regulate the obligations of public authorities and individuals regarding protection against and coping with specific problematic health areas, such as infectious diseases (Ustawa z, 6 September 2001, r. o chorobach zakaźnych i zakażeniach, Dz.U. Nr 126 z 2001 r. poz.1384 z późn.zm), mental health (Ustawa, 19 August 1994, r. o ochronie zdrowia psychicznego, Dz. U. Nr 111, poz. 535, z późn. zm.), drug (Ustawa, 24 April 1997, r. o przeciwdziałaniu narkomanii, Dz. U. z 2003 r. Nr 24, poz. 198 i Nr 122, poz. 1143), and alcohol abuse (Ustawa, 26 October 1982, r. o wychowaniu w trzeźwości i przeciwdziałaniu alkoholizmowi, Dz. U. z 2002 r. Nr 147, poz. 1231, z późn. zm.10) problems-which.Laws on the rescue system (Ustawa, 25 July 2001, r. o Państwowym Ratownictwie Medycznym, Dz. U. Nr 113, poz. 1207, z późn. zm.13) and services (Ustawa, 6 December 2002, r. o świadczeniu usług ratownictwa medycznego, Dz. U. Nr 241, poz. 2073 oraz z 2003 r. Nr 99, poz. 920) which regulate the functioning and financing of the national rescue system and care provided to persons in need.Social security law which establishes rules for social security coverage for various population groups, both occupationally active and nonactive (Ustawa, 13 October 1998, o systemie ubezpieczeń społecznych, Dz. U. Nr 137, poz. 887 z późn. zm.).Penal code which regulates the issues of crime and treatment of persons convicted of committing crimes (Ustawa, 6 January 1997, r.-Kodeks karny wykonawczy, Dz. U. Nr 90, poz. 557, z późn. zm.9).Law on foreigners which regulates the rights and obligations of non-Polish residents who are under care of Polish authorities (Ustawa, 13 January 2003, r. o cudzoziemcach, Dz. U. Nr 128, poz. 1175 oraz z 2004 r. Nr 96, poz. 959 i Nr 179, poz. 1842).Road traffic code which regulates the rights and obligations of public roads users (Ustawa, 20 January 1997, r. o ruchu drogowym, Dz.U. Nr 58 z 2003 r. poz.515 z późn.zm.).
Many of these laws contain so-called delegations to regulations/decrees regulating on a technical level both the scope and conditions of services available to individuals in Poland. The underlying logic of the legislative process in Poland provides that laws establish the fundamental principles and mechanisms while regulations/decrees govern their practical application. Both are by nature obligatory, and there are legal and administrative sanctions for noncompliance. In addition to legal acts, there are additional documents referring to the benefit basket which are not of legal nature. The most important of these and the only one discussed here is a catalogue of services (so-called “contracted products”) which are purchased by the NHF. The catalogues are appendices to the procurement documentation in the procedures of purchasing health services and goods within the universal health insurance system. The “products” included in the catalogues are then the subject of contracts between the NHF and the service providers, to be provided to the “beneficiaries.”
Table 1 presents a list of laws and decrees which determine the benefit basket in each of the existing subsystems. The level of detail in defining the scope of the benefits to which persons are entitled, differs in the Acts. It is worth noting that in each case the Act states who is entitled (covered) to a certain kind of protection and what protection it is, sometimes also defining the circumstances and conditions under which it is available. For present purposes we can differentiate three levels of explicitness of definitions in the benefit basket:Table 1 List of documents determining the benefit basket and its level of explicitnessExplicitSemiexplicitImplicit1. Health insurance law+++  Decree on dental care+––  Decree on basic and supplementary pharmaceuticals+––  Decree on pharmaceuticals for chronically ill+––  Decree on prevention services+––  Decree on prevention services, school medicine+––  Decree on medical good and materials+––  Decree on medical transportation benefits+––  Decree on spa therapy services–+–  Decree on highly specialized procedures+––  Services procurement documentation+––2. Mental health protection law––+3. Social insurance law––+  Decree on rehabilitation measures–+4. Farmers social insurance law––+  Missing decree–––5. Penal code–+–6. Alcohol abuse law–+–7. Drugs abuse law–+–8. Law on foreigners––+9. National rescue system law––+10. Rescue services law––+11. Labor code––+  Decree on prevention measures for employees–+–  Decree on prevention measures for self-employed and others–+–12. Occupational medicine law–+–  Decree on occupational medicine in defense services+––  Decree on occupational medicine in internal affairs services+––  Decree on occupational medicine in penitentiary services+––  Decree on occupational medicine in National Railways+––13. Infectious diseases law–+–  Decree on immunization of employees+––  Decree on obligatory immunizations+––14. Road traffic law–+–  Decree on obligatory drivers health tests+––Explicit: That which is understood as providing specific medical procedures and actions (e.g., immunization for hepatitis B), namely those mentioned in the law or regulationSemiexplicit: That which outlines a certain area of health care to which beneficiaries have rights (e.g., primary care)Implicit: That which mentions generally that health care is provided, sometimes with additional formulation as all necessary, efficient, etc.In some of the Acts included in Table 1 some benefits are determined in more detail and some others in less detail, which means that the level of explicitness of defining the benefit basket ranges from implicit to explicit. As a rule, regulations (decrees) are more detailed in the benefits definition than laws, and the laws are more specific than the Constitution. However, in the case of the most prominent Act, the Universal Insurance Law, the Act encloses the benefits definition on all three levels of detail.
Obligations of the public authorities derived from the legal regulations are financed from public sources, which amount to 70% of the total health expenditure in Poland. Private expenditure (estimated as 30% of the total health expenditure) is mainly out-of-pocket payment with minor supplemental role of private insurance and quasi-insurance. As regards private expenditure, no benefit basket can be discussed; the only exception is occupational medicine, discussed below.
Roles of partners in defining the benefit basket
Parliament is the supreme legislative body in Poland. The central government, respecting (in theory) a subsidiary principle, regulates the activities of both individuals and institutions and, through its administration and agencies supervises their compliance with legal regulations. Local (regional) governments are responsible for public activities on their territories. Laws establish principles and mechanisms, while regulations/decrees establish their practical applications. Regulations/decrees cannot exceed the area determined by the specific law, and they are issued by each of the government ministries. With regards to the benefit basket, the National Assembly, i.e., a joint session of Sejm and Senate (lower and the upper chambers of Parliament), adopts the Constitution, among others, with its provisions dedicated to health protection. The Sejm drafts laws that must be approved by the Senate and signed by the President. The Government ministries define various parts of the “benefit basket” issuing regulations to existing legal provisions.
The NHF defines catalogues of services (so-called “products”) which are purchased by the health insurance institution that implements universal mandatory health insurance. The process of defining the list of products was not formalized until recently, although in 2005 a formalized process of setting general conditions of contracting was established, which take places under the NHF protectorate and with participation of providers associations representing more the 10,000 professionals. The catalogues are published as appendices to the procurement documentation in the process of purchasing health services and goods within the universal health insurance system. The “products” from catalogues are later placed in contracts between the NHF and the providers of the services.
Definitions of the benefit basket in certain areas of health care
Health care is a wide area, and sometimes it is difficult to define which actions belong to it and which, for example, to social security. For the present study, the OECD functional classification (International Classification for Health Accounts–Health Functions) was used to define boundaries of the health care system. Table 2 presents areas/subsystems of health care in Poland with their respective regulations regarding the benefit basket and their characteristics; the individual Acts presented in Table 1 are aggregated here into following areas:Table 2 Benefit basket and an explicitness of its description in distinguished areas (L law, R regulation,
A nonlegal document,1 least precise, 3 most precise)Social health insurance systemSocial insurance systemOccupational medicineRescue systemMental health and substance abuseInfectious diseasesPrisoners, foreigners under state careLRALRALRALRALRALRALRAHC.1 Services of curative care––––––––––––––––––1––HC.1.1 Inpatient curative care233––––––2–31––1–3–––HC.1.2 Day cases of curative care1–3–––––––––1––––––––HC.1.3 Outpatient care2–3–––––––––1––1–3–––HC.1.3.1 Basic medical and diagnostic services2–3–––––––––1––1–––––HC.1.3.2 Outpatient dental care233–––––––––1––1–3–––HC.1.3.3 All other specialized health care2–3–––––––––1––––––––HC.1.3.9 All other outpatient curative care2–3–––––––––1––––––––HC.1.4 Services of curative home care2–3–––––––––––––––––HC.2 Services of rehabilitative care––––––––––––––––––1––HC.2.1 Inpatient rehabilitative care1–2123––––––1–2––––––HC.2.2 Day cases of rehabilitative care1–3–––––––––1–2––––––HC.2.3 Outpatient rehabilitative care1–31–3––––––1–2––––––HC.2.4 Services of rehabilitative home care1–3–––––––––1–2––––––HC.3 Services of long-term nursing care–––––––––––––––––––––HC.3.1 Inpatient long-term nursing care2–3–––––––––1––––––––HC.3.2 Day cases of long-term nursing care1–3–––––––––1––––––––HC.3.3 Long-term nursing care: home care2–3–––––––––1––––––––HC.4 Ancillary services to health care––––––––––––––––––1––HC.4.1 Clinical laboratory2–3–––––––––1––2–3–––HC.4.2 Diagnostic imaging2–3–––––––––1––2–3–––HC.4.3 Patient transport and emergency rescue222––3–––1–21–––––1––HC.4.9 All other miscellaneous services–––––––––––––––––––––HC.5 Medical goods dispensed to outpatients––––––––––––––––––1––HC.5.1 Pharmaceuticals and medical nondurables––––––––––––1––––––––HC.5.1.1 Prescribed medicines23––––––––––1––23––2–HC.5.2 Therapeutic devices and medical durables–––––––––––––––––––––HC.5.2.1 Glasses and vision products23–––––––––––––––––––HC.5.2.2 Orthopedic devices and other prosthetics23–––––––––––––––––––HC.5.2.3 Hearing aids23–––––––––––––––––––HC.5.2.4 Medicotechnical devices, incl. wheelchairs23–––––––––––––––––––HC.5.2.9 All other miscellaneous medical durables23–––––––––––––––––––HC.6 Prevention and public health services––––––––––––––––––1––HC.6.1 Maternal and child health223–––––––––1––2–3–––HC.6.2 School health services223––––––––––––2–3–––HC.6.3 Prevention of communicable diseases123––––––––––––233–––HC.6.4 Prevention of noncommunicable diseases123–––––––––1––––––––HC.6.5 Occupational health care––––––12–––––––––––––HC.6.9 All other miscellaneous public health services––––––––––––1––––––––Social health insurance system, regulated by health insurance law and related decrees. Procurement documentation belongs to this system, which is not a legal act but is discussed here because of its role in the system.Social insurance system, which is a wider concept than social health insurance, but here it contains functions regulated by social insurance law and farmers’ social insurance law (Ustawa z dnia 20 grudnia 1990 o ubezpieczeniu społecznym rolników i ich rodzin, Dz.U. z 1998r. Nr 7 poz.25 z późn.zm.) and related decrees.Occupational medicine, which is a privately funded system, according to OECD definitions, often in the form of prepaid arrangements of employers, covering prevention services and often some forms of outpatient health care.Rescue system, comprising benefits resulting from regulations on national rescue system and rescue services.Mental health and substance abuse, comprising benefits for persons suffering from mental disorders and substance abuse problems.Infectious diseases, covering prevention and treatment services, partly provided on a mandatory basis.Prisoners and foreigners residing in closed centers, covering all necessary medical interventions for persons with limited freedom.Table 2 describes the kind of document that regulates the benefit basket in each subsystem and the level of precision in determining the basket, which refers to the distinctions mentioned above (1 the least precise, 3 the most precise).
Social health insurance system
Most health services and goods are delivered within the social health insurance system, which we therefore discuss in more detail. After lengthy debate and two major changes in the law in 1997 and 2003 and the Law of 27/08/2004 on health care services funded from public resources was adopted in 2004 to replace existing health insurance laws (Letter of Issue no. 210, 2004, pos. 2135). Under the new law the beneficiaries, i.e., EU citizens with insurance rights, gained the right to access the entire range of available health care services. Publicly funded services are divided into 22 different categories, for example, diagnostic tests including medical laboratory diagnostic, services aimed at health protection, prevention of diseases and injuries, early detection of diseases, including compulsory vaccinations, primary health care, outpatient specialist services, and inpatient care. The Minister of Health has several legal obligations for issuing the regulations partially forming the benefit basket. These are specified in Table 1.
According to Article 16 of the Act on health care services financed from public funds, the benefit basket does not cover some services, such as reimbursement of the cost of certificates required for a driving license and other medical statements or forensic expertise obtained at the request of the court or prosecutor, which are covered by separate public funds. Patients residing at nursing homes must cover the cost of food and accommodation themselves. There is a negative list of services excluded from the guaranteed public health service basket, such as vaccinations not included in the compulsory vaccination package, plastic and cosmetic surgery when it is not necessary treatment for malformation, injury, illness, and gender change operations but also treatment of epilepsy with stimulation of nervus vagus, diagnostics and treatment of the taste disorders, hyperbaric chamber in SM treatment. A full list of these services was appended to the law, which means that it must be ratified by Parliament. Moreover, according to Article 25 of the Act, the NHF does not reimburse the costs of treatment or diagnostics of an insured person that are incurred abroad unless these services were provided in accordance with Regulation 1408/71/EC. On the basis of these regulations and previously presented legal provisions the NHF defines the so-called “contracted products” that are purchased in the scheme. The catalogue of health care products includes a number of parts/chapters, as presented in Table 3.Table 3 Categories of “contracted products” purchased by National Health Fund with classification by SHA categories and short description, valid for 2005SHA categoryCharacteristicsPrimary careHC.1.3.1, HC.6.1, HC.6.2, HC.6.3Provided by general practitioners, pediatricians, and internal medicine specialists. The care is supported by general-practitioner nurses and midwives. Contains also school nurse activityOutpatient care, surgicalHC.1.3.3Contains approx. 50 invasive (surgical) procedures provided by outpatient care physiciansOutpatient care, medical and diagnosticsHC.1.3.3Contains consultation services of outpatient care physicians, together with a set of diagnostic tests that must be provided by them at their own costOutpatient care, diagnosticsHC.4.2Contains 22 imaging procedures financed separatelyHospital care, generalHC.1.1Contains approx. 1400 different hospital care cases; both surgical (with invasive procedure) and medical (conservative therapy)Hospital care-radiology, nuclear medicineHC.1.1, HC.1.2Contains nuclear medicine therapies, provided with or without hospitalizationHospital care, oncological therapyHC.1.1, HC.1.2Contains chemotherapy of neoplasm, both in hospital care and day careHospital care, nononcological therapyHC.1.1, HC.1.2Contains chemotherapy for nononcological cases (e.g., hepatitis B), provided both in hospital and day careDentistry, childrenHC.1.3.2Contains approx. 40 procedures available to children (up to 18 years) onlyDentistry, generalHC.1.3.2Contains approx. 70 procedures available to all insurersDentistry, pregnant womenHC.1.3.2Contains services that are available for free to pregnant womenSpasHC.2.1, HC.2.3Contains rehabilitative and treatment services available in spas (a category of settings), both in inpatient and ambulatory mannerEmergencyHC.4.3Contains rescue and emergency transportation services, both air and groundSeparately contracted servicesHC.1.2Contains approx. 20 not otherwise classified services, mainly provided in day care (incl. hemodialysis, oxygen therapy, etc.)Psychiatric and substance abuse therapyHC.1.1, HC.1.3.3, HC.1.2Contains services provided by psychiatrists, in all kind of settingsRehabilitationHC.2.1, HC.2.2, HC.2.3, HC.2.4Contains rehabilitation services provided by physicians and physiotherapists, in all kinds of settingsLong-term careHC.3.1, HC.3.2, HC.3.3Contains long- term nursing care, but also palliative care and hospices, in all kinds of settingsHealth promotion and preventionHC.6.4Contains prevention and early detection programs for noncommunicable diseases
The catalogue of services is not published in a uniformed way but rather as a set of appendices to various models of contracts with various groups of providers. It is published in electronic form usually in October of the year preceding the year of contracting. Table 3 was developed on a basis of an official publication of the NHF and presents an aggregate of a product list from a variety of NHF internal documents. The logic of products nomenclature reflects the way in which it is used for payment and monitoring purposes. Some parts of the catalogue are published in the form of legal acts and appendices and are rewritten into contractual conditions, for example, dentistry, spas, and medical products (including orthopedic) while others are developed fully by the NHF experts.
Other schemes
A number of laws regulate the other schemes providing services and goods to Polish inhabitants (see Table 1). General characteristics of the remaining schemes are that they usually rely on public funds and implement social and public policy in delivering services and goods to various population groups. Some of the schemes obligatorily engage private funds, for example, occupational medicine which obliges employers to provide certain kind of services to employees. The special regulations often focus on underprivileged populations, such as the mentally ill and alcohol and drugs addicts, to make available necessary services to persons who would otherwise not receive them. It is interesting to note that many of the functions specified in these special regulations oblige the NHF to finance it, as in the case of infectious diseases.
Discussion
This study presents the legal and administrative regulations regarding health benefits to which various groups in Poland are entitled. The range of available benefits is clearly quite wide, and the justifications for respective entitlements are also varied. However, in both public and professional opinion there is no such a thing as the “benefit basket” in Poland [1]. This is also frequently the reason adduced to explain why the health system works so poorly. There is no systematic research in this area, but the different parties in the system appeal for the provision of a benefit basket clearly for different reasons: patients who feel lost in the system see in the envisioned benefit basket a way to secure their rights, and professionals have claimed that the lack of explicit listing of benefits make patients demand more then necessary, which places physicians under strong pressure to prescribe and perform many services that are not eventually covered by the payer. On the other hand, explicit formulation of “products” purchased in the insurance system creates substantial controversies and criticism because some medical procedures are omitted.
In 1999–2000 the Ministry of Health and Social Welfare publicly expressed its commitment to the development of a defined basic benefit basket. This proposal was hotly disputed at the time, with a number of groups opposing the concept because it was often seen as a way of limiting available health care and thus saving money. The debate prompted the Ministry to seek outside assistance in the form of consultants’ advice. The consultation project was aimed at “verifying and arranging the range of services purchased by the Health Funds” regarding determination of the range of guaranteed services, the so-called basic benefit basket, with specification and terms of their provision [2]. The formulated range and description of services were expected to serve as the basis for the Health Funds in drafting contracts with service providers. It was also intended to serve as a tool for performance assessment of particular Sickness Funds, made by the Health Insurance Supervisory Office and the Ministry of Health.
In January 2004 the Polish Constitutional Tribunal announced its finding (Trybunał Konstytucyjny orzeczenie, 7 January 2004, r. (Sygn. Akt K 14/03) that the existing health insurance law was unconstitutional because it failed to define with sufficient precision the scope of services that beneficiaries may expect from the national health system as mentioned in the Constitution (“Conditions and scope of services will be detailed in the appropriate law,” Paragraph 62). The Government was given approx. 1 year to prepare a new law to fulfill this requirement.
In formulating the new law many different possibilities were considered. One suggestion was that the law should contain a list of medical procedures which are performed in the system. This idea was considered unrealistic, however, especially because of the short period available for drafting the law. There were strong voices to the effect that some one must distinguish clearly between those services that are provided fully free of charge within the system and those that must be paid for either partially or in full by the patients. This differentiation was made by a group of experts assembled by the Minister of Health [3]. The concept also considered the establishment of a special institution, which would make technology assessments of medical interventions and participate in the decision-making process on inclusion and exclusion of the interventions in the benefit basket for universal health insurance. Finally, because there was no prevailing opinion as to the final role of the technology assessment institution, it was not established. However, the Minister of Health was required by stipulations in the last paragraph of the insurance law to “undertake efforts to establish an organizational structure, whose task would be to make health technology assessment of medical procedures, with special regard to procedures, which were subject to purchasing by the Fund.” Certain changes in insurance law, namely attachment of a list of excluded procedures, together with a promise to establish an institution of health technology assessment, solved the legal controversy between Parliament and Constitutional Tribunal.

poland
health services
health benefit plans
health priorities
national health programs


Feasibility, reliability, and validity of adolescent health status measurement by the Child Health Questionnaire Child Form (CHQ-CF): internet administration compared with the standard paper version

Aims In this study we evaluated indicators of the feasibility, reliability, and validity of the Child Health Questionnaire-Child Form (CHQ-CF). We compared the results in a subgroup of adolescents who completed the standard paper version of the CHQ-CF with the results in another subgroup of adolescents who completed an internet version, i.e., an online, web-based CHQ-CF questionnaire.
Introduction
During the past two decades, several measures have become available to describe generic health-related quality of life in pediatrics, but adolescent self-report questionnaires received relatively little attention until now [1, 2]. The Child Health Questionnaire (CHQ) is one of the most widely used pediatric health-related quality of life measures and has been translated into 21  languages (32  countries). There is a form for parents and also a self-report form for adolescents, the Child Health Questionnaire-Child Form (CHQ-CF) [2–7]. The CHQ covers physical and psychosocial aspects of health, and includes the impact of child health problems or handicaps on family life [3]. This study focuses on the evaluation of missing answers at the item level, distribution of the scale scores, reliability, and validity of the CHQ-CF in an adolescent population.
We expect the commonly used paper format of the CHQ and other health questionnaires to be increasingly replaced by internet versions, especially in adolescent populations that are accustomed to the use of computers and the internet [8]. From the perspective of clinicians and researchers, the advantages of using the internet include avoiding paper work, on-line data-entry, and procedures designed to reduce the number of missing answers and the length of questionnaires [9, 10].
In general, the mode of questionnaire administration (e.g., written questionnaire, face to face interview, telephone interview, computer questionnaire) may affect the participation rate, number of missing answers, psychometric properties, and actual scores [11–14]. With regard to health questionnaires, several studies demonstrated some differences between the commonly used paper versions and computer versions of the same questionnaires [15–17]. Especially in studies comparing paper and computer questionnaires on sensitive topics, administration via computer was found to increase reporting of e.g., drug use or unsafe sexual behaviors, as this medium is apparently perceived as providing more privacy than a paper form [18–20].
With regard to online, i.e., internet or web-based administration of health questionnaires, several studies have demonstrated that online health questionnaires are feasible in various settings, especially among adolescents [21, 22]. However, very few randomized studies have evaluated whether psychometric properties and scores differ between the paper and the internet mode of administration of the same health questionnaire [23–26].
In this study, we compared indicators of the feasibility, reliability, and validity of the CHQ-CF in a subgroup of adolescents who completed the standard paper version of the CHQ-CF with the same indicators in another subgroup of adolescents who completed a newly developed internet version of the questionnaire. Additionally, we compared the mean CHQ-CF scores and distributions of the scale scores between both subgroups. A randomized parallel group design was applied in a large adolescent population (13–7  years old), ensuring that both subgroups were comparable.
The study assessed and compared the paper and internet mode of CHQ-CF administration with regard to the following indicators:
the number of missing answers (indicator of feasibility),the distribution of the scale scores including mean scale scores in the whole sample and in gender and age specific subgroups,the internal consistency reliability of multi-item scales (indicator of reliability),the ability of the CHQ-CF to discriminate between subgroups with and without self-reported chronic conditions (indicator of construct validity).
Methods
Study population
In 2003, 1,071  students in 55  classes of various educational levels in the 3rd year of seven secondary schools (13–7 years old) in the area of Vlaardingen (metropolitan area) and Harderwijk (rural area), The Netherlands, were invited to complete the Child Health Questionnaire Child Form (CHQ-CF). The parents and students each received written information about the study several weeks before data collection; parents could refuse their child’s participation, and participation by the students was voluntary.
Data collection
The CHQ-CF consists of 87  items with 4, 5, or 6 response options divided over 10 multi-item scales and two single-item scales (Table 1) [3]. To reduce respondent burden, the item “change-in-health–was not fielded in this study, and the CHQ-CF scales “role functioning-emotional–and “role functioning-behavioral–were combined into a single scale. The combination of the two role functioning scales is a departure from the CHQ-CF instructions that makes the test analogous to the parent form of the CHQ in this regard [3]. For each scale, items were summed up (some recoded/recalibrated) and transformed into a 0 (worst possible score) to 100 (best possible score) scale [3]. Items on standard socio-demographic variables and the prevalence of seven chronic conditions were included in the questionnaire. From the conventional paper format, using the same wording of the items and instructions, an internet version of the questionnaire was developed through a generic internet tool using PHP (4.0.1), MySQL (3.22), and JavaScript (1.3) [27]. The internet version of the questionnaire listed the items of each CHQ-CF scale on a separate web-page. The internet version did not allow the respondent to select more than one answer to each item of the CHQ-CF and it checked the questionnaire for missing answers before the respondent could “logout– If one or more of the items were not answered, the internet version prompted the respondent to go back to complete those items; but, if the user failed to “logout–properly, missing answers would remain.Table 1CHQ-CF scales, items per scale, and interpretation of low and high scoresaCHQ-CF ScalesNumber of itemsDescription low scoreDescription high scorePhysical functioning (PF)9Child is greatly limited in performing all physical activities, including self-care, due to healthChild performs all types of physical activities, including the most vigorous without limitations due to healthRole functioning: Emotional (RE)b3Child is greatly limited in school work or activities with friends as a result of emotional problemsChild has no limitations in schoolwork or activities with friends as a result of emotional problemRole functioning: Behavioral (RB)b3Child is greatly limited in school work or activities with friends as a result of behavior problemsChild has no limitations in schoolwork or activities with friends as a result of behavior problemsRole functioning: Physical (RP)3Child is greatly limited in school work or activities with friends as a result of physical healthChild has no limitations in schoolwork or activities with friends as a result of physical healthBodily pain (BP)2Child has extremely severe, frequent and limiting bodily painChild has no pain or limitations due to painGeneral behavior (BE)17Child very often exhibits aggressive, immature, delinquent behaviorChild never exhibits aggressive, immature, delinquent behaviorMental health (MH)16Child has feelings of anxiety and depression all of the timeChild feels peaceful, happy, and calm all of the timeSelf esteem (SE)14Child is very dissatisfied with abilities, looks, family/peer relationships and life overallChild is very satisfied with looks, family/peer relationships and life abilities, overallGeneral health perceptions (GH)12Child believes his/her health is poor and likely to get worseChild believes his/her health is excellent and will continue to be soChange in health (CH)c1Child’s health is much worse now than 1  year agoChild’s health is much better now than 1  year agoFamily activities (FA)6The child’s health very often limits and interrupts family activities or is a source of family tension.The child’s health never limits or interrupts family activities nor is family a source of tensionFamily cohesion (FC)1Family’s ability to get along is rated “poor–Family’s ability to get along is rated “excellent–aReproduced with permission [3]bThe CHQ-CF scales “Role functioning-emotional–and “Role functioning-behavioral–were merged into a single scale “Role functioning-emotional/behavioral–(REB) in this studyc This single-item scale was not fielded in this study
Randomization
Within each school class, students were randomly assigned to either the paper or the internet mode of administration using SPSS-generated random numbers. Students completed the questionnaires, either on paper or online in a classroom with computers linked to the internet, under the supervision of a research assistant; the students were allowed adequate privacy.
Analysis
Preparatory secondary vocational education was labelled as “lower secondary education”; secondary schools that prepare students for higher professional training as "intermediate secondary education", and university preparatory secondary education as "higher secondary education". Differences between the characteristics of the participants allocated to the paper versus the internet versions of the questionnaires were tested by Student’s t-test and the χ2 test. We assessed the frequency of missing answers to CHQ-CF items; the difference in the number of missing answers between the two formats was assessed by the Mann-Whitney U test. We assessed the distributions of the CHQ-CF scale scores to evaluate floor and ceiling effects (≥25% of the respondents having the lowest/highest score) for both modes of administration. Differences between CHQ-CF scale scores by format in the total sample were assessed by Mann-Whitney U tests. Additionally, after transforming the scale scores into ranks, ANOVA was applied to test whether the mode of questionnaire administration interacted with the variables gender (male n = 432; female n = 501) and age (13–4  year olds, n = 399; 15–7  year olds, n = 534). Cohen’s effect sizes, defined as d = [Mean(a) Mean(b)]/SD, where the denominator was the square root of [(na-1)SDa2 + (nb-1)SDb2] / [(na-1) + (nb-1)], were applied to indicate the relative magnitude of score differences between modes of administration. Here, the letters "a" and "b" refer to the subgroups administered the paper and internet forms of the test, respectively [28]. Following Cohen’s suggested guidelines, 0.20 ≤d <  0.50 indicated a “small effect– 0.50 ≤d <  0.80 a “medium effect– and d ≥0.80 a “large effect–[28]; Norman et al. have suggested that, in general, d = 0.50 can be considered as threshold for a “minimally important difference–(MID) [29]. Cronbach’s α was applied to evaluate the internal consistency reliability of CHQ-CF multi-item scales by format; α of 0.70 or higher was considered to indicate sufficient internal consistency reliability [30]. We applied statistical tests of the hypothesis that the Cronbach’s α reliability coefficients of CHQ multi-item scales in the sample administered the test on paper were equal to those administered the test online [31]. We applied item-level discriminant tests to evaluate whether the CHQ-CF items represent separate domains. For each mode of questionnaire administration, we evaluated whether (on average) correlation coefficients (Pearson-r correlation coefficients) between the items and their own scale score (without the item under consideration) were higher than the correlation coefficients between these items and any other scale. The average Pearson-r correlation coefficients were calculated by applying Fisher’s z transformations [32]; we tested whether the differences between the average Pearson-r correlation coefficients in the subgroup administered the paper form and in the subgroup administered the test online were statistically significant [33]. We assessed the CHQ-CF’s ability to discriminate between subgroups with 0, 1 or 2, and 3 or more chronic conditions, after having transformed the CHQ-CF scale scores into ranks, by ANOVA with the independent variables “number of chronic conditions– "mode of questionnaire administration– and the interaction term “number of chronic conditions–“mode of questionnaire administration– Cohen’s effect sizes d = [Mean(a) Mean(b)]/SD in the condition subgroup were calculated for 1 or 2 versus 0 conditions, and for ≥3 versus 0 conditions. The designations "a" and "b" refer to the subgroups without chronic conditions and those with chronic conditions, respectively [28].
All analyses were done using SPSS, Version 11.0.1. The medical ethical committee of the Erasmus MC-University Medical Center Rotterdam, approved the study.
Results
Participants and randomization
The participation rate was 87%. The age range of the participants was 13–7  years (mean age 14.7  years; SD 0.68), 54% were female, 93% were born in the Netherlands, and the majority attended lower secondary education (Table 2). The prevalence of self-reported chronic conditions was as follows: asthma, 8%; allergies, 25%; hearing problems, 7%; visual problems, 8%; headaches or migraine, 17%; chronic lower back pain, 17%; and depression or anxiety attacks, 8% (Table 2). These characteristics were equally distributed in the groups assigned to the paper and internet versions of the questionnaires (P ≥0.05; Table 2). The demographic characteristics of the participants (age, gender, country of birth, and educational level) reflected those of the general population of Dutch adolescents [34].Table 2Characteristics of study participants (total sample: n = 933; participants assigned to the paper questionnaire:n = 475; participants assigned to the internet questionnaire: n = 458)Total study group (n = 933)Group with paper mode of administration (n = 458)Group with Internet administration (n = 475)P-valueMean (SD) or Rangen% of ParticipantsMean (SD) or Rangen% of ParticipantsMean (SD) or Rangen% of ParticipantsInternet versus paper modeDemographic characteristicsAge (years)Mean (SD)14.7 (0.68)14.7 (0.68)14.7 (0.68)0.61aRange13–713–713–7GenderWomen50154%24451%25756%0.17bBorn in the NetherlandsYes86693%44193%42593%0.90bEducational level of the schoolLower secondary education54558%27458%27159%Intermediate secondary education17919%9420%8519%0.88cHigher secondary education20922%10723%10222%Chronic conditions:AsthmaYes768%408%368%0.81bAllergiesYes22925%11825%11124%0.88bProblems with hearingYes627%306%327%0.70bProblems with seeingYes788%398%399%0.91bHeadaches or migraineYes15917%8117%7817%1.00bChronic low back painYes15917%8819%7116%0.22bDepression and/or anxiety attacksYes748%368%388%0.72ba Student’s t-testb Chi square test df = 1c Chi square test df = 2
Difference in the number of missing answers between different modes of CHQ-CF administration
At the item level, use of the paper version of the CHQ-CF resulted in more missing answers (0–.89% per item; mean 0.54%) compared with the internet version (0–.22% per item; mean 0.04%; P <  0.01).
CHQ-CF scores by mode of administration
A ceiling effect was observed for four CHQ-CF scales in the subgroup that completed the paper questionnaire, and 3 CHQ-CF scales in the subgroup that completed the internet questionnaire (Table 3). Four CHQ-CF scales, i.e., “general behavior– “role functioning-physical– “mental health– and “family activities– resulted in statistically significant, higher scores for paper versus internet administration (P <  0.05), but the effect sizes (d) were ≤0.21 (Table 3). The mode of questionnaire administration did not interact significantly with gender (P ≥0.05 regarding all scales), nor with age (P ≥0.05 regarding six scales), except for the CHQ-CF scales “role functioning-emotional/behavioral–(P <  0.05), “mental health–(P <  0.05), “self esteem–(P <  0.05), and “general health–(P <  0.01). Regarding these 4 CHQ-CF scales, administration of the paper version resulted in lower scores than online administration (or nearly equal scores in the case of “mental health– in the subgroup of 13–4  year olds, while in the subgroup of 15–7  year olds, paper administration resulted in higher scores compared with internet administration; the Cohen’s effect sizes (d) for these differences, regardless of sign, were ≤0.21 (data not shown).Table 3Comparison of mean scores, distributions of the scale scores, and other psychometric properties of CHQ-CF scales in subgroups with paper (n= 475) and internet modes (n = 458) of questionnaire administrationCHQ-CF scalesa (range 0–00)Mode of admini strationMean (SD)Paper versus internet mode of administrationRange of scores% maxd% mine25th %tile50th%tile75th%tileCronbach’s alphaAverage item-own scale correlation fAverage item-other scale correlationP-value (MWU)bEffect size dcPhysical FunctioningPaper96.0 (6.9)0.370.0144–00560961001000.690.400.19Internet95.8 (7.2)37–00530931001000.720.440.18Role funct.-Emo/behPaper89.4 (17.2)0.140.000–00600781001000.810.650.35Internet89.5 (15.1)0–00540861001000.70 g0.51 g0.28Role funct.-PhysicalPaper95.0 (12.9)0.020.0522–008101001001000.860.740.30Internet94.4 (11.7)22–00750891001000.76 g0.60 g0.25Bodily PainPaper73.5 (22.7)0.56–0.050–0025060801000.880.800.35Internet74.7 (21.4)0–002416080900.890.810.30General BehaviorPaper80.9 (10.6)0.000.2125–00107582880.830.440.26Internet78.6 (11.4)35–00007279870.850.460.26Mental HealthPaper76.5 (15.4)0.010.1214–00206980890.920.630.35Internet74.6 (14.8)14–00006777840.90 h0.600.31Self EsteemPaper74.7 (12.2)0.990.0220–00106875820.880.560.30Internet74.4 (11.6)25–00007076820.870.550.28General HealthPaper73.5 (16.5)0.590.0519–00306376860.820.500.28Internet72.6 (16.7)20–00106375860.810.580.28Family ActivitiesPaper80.0 (17.7)0.030.118–001706783960.810.580.24Internet78.1 (16.9)25–001306779920.780.540.24Family CohesionPaper70.6 (23.5)0.910.000–00182608585nana0.36Internet70.7 (23.0)0–00162608585nana0.33aThe CHQ-CF scales ‘Role functioning-emotional–and ‘Role functioning-behavioral–were merged into a single scale in this studybTwo-sided Mann-Whitney U test of scale scores of the group using paper, and the group using the internet version of the questionnairecDifference of mean scores divided by weighted average of SDs in groups given the paper and internet version of the questionnaires [28, 29]d/e% of respondents with best/worst possible score (ceiling/floor)fEach item was correlated with the applicable ad-hoc scale without the item under considerationg/hStatistically significant differences of Cronbach’s alpha/average Pearson-r correlation coefficients in subgroups given paper or internet questionnaires (gP ≤0.01; hP ≤0.05) [31, 33]. na Not applicable; Role funct.-Emo/beh - Role functioning-emotional behavioral; Role funct.-Physical - Role functioning-physical
Internal consistency reliability of scales by mode of administration
Cronbach’s αs for the two formats were adequate for all CHQ-CF scales, except “physical functioning–in the subgroup administered the paper version of the questionnaire (α =  0.69). The two “role functioning–scales and “mental health–showed statistically significant, higher Cronbach’s αs in the subgroup administered the paper version of the questionnaire compared with the alphas in the subgroup administered the internet version (P <  0.01, respectively P <  0.05) (Table 3). All multi-item scales, regarding both modes of administration, showed higher average (corrected) item-own scale correlation coefficients than average item-other-scale correlation coefficients. The two “role functioning–scales showed statistically significant, higher average item-own scale correlation coefficients in the subgroup administered the paper version of the questionnaire compared with the item-own scale correlation coefficients in the subgroup administered the internet version (P <  0.01) (Table 3).
Construct validity by mode of administration
All mean CHQ-CF scale scores were lower in the subgroup with one or two reported conditions and in the subgroup with three or more reported conditions when either was compared with the subgroup with no reported conditions. For both modes of questionnaire administration, and for all CHQ-CF scales, the more chronic conditions that were reported, the higher the effect sizes compared with the subgroup with no chronic conditions. ANOVA showed statistically significant CHQ-CF score differences by “number of chronic conditions–for all scales (P <  0.01) (Table 4). The mode of questionnaire administration did not interact significantly with the variable “number of chronic conditions–(P ≥0.05 for all scales) (Table 4).Table 4Ability of the CHQ-CF scales to discriminate between subgroups differing in the participants–number of chronic conditions, for the group that was assigned to complete the paper version (n = 475) and for the group that was assigned to complete the internet version (n = 458)Number of chronic conditions per participantCHQ-CF scalesa (range 0–00): Mode of admini- stration0 conditions1 or 2 conditions≥ conditions1 or 2 versus 0 conditions≥ versus 0 conditionsANOVA P-value Number of chronic conditionsANOVA P-value Internet versus paper modeANOVA P-value Interaction term Number of chronic conditions by mode of questionnaire administrationn = 224 (Paper)n = 206 (Internet) Mean (SD)n = 210 (Paper)n = 217 (Internet) Mean (SD)n = 41 (Paper)n = 35 (Internet) Mean (SD)Effect size dbEffect size dbPhysical functioningPaper97 (5)96 (7)90 (10)0.240.80*Internet97 (5)95 (7)89 (12)0.280.64*0.000.950.52Role funct.-emo/behavPaper93 (13)88 (18)77 (24)0.250.64*Internet91 (14)89 (15)79 (18)0.150.66*0.000.200.72Role funct.-physicalPaper97 (11)94 (13)91 (17)0.190.34Internet96 (10)94 (13)91 (13)0.140.340.000.070.69Bodily painPaper82 (18)70 (22)47 (24)0.52*1.45*Internet80 (18)72 (21)55 (24)0.391.03*0.000.200.32General behaviorPaper84 (9)80 (10)71 (13)0.400.98*Internet81 (10)78 (11)69 (14)0.260.90*0.000.030.65Mental healthPaper83 (10)74 (16)57 (16)0.56*1.58*Internet80 (11)72 (15)59 (18)0.501.20*0.000.260.29Self esteemPaper79 (10)73 (12)61 (12)0.52*1.44*Internet77 (9)73 (12)66 (14)0.330.80*0.000.260.06General healthPaper80 (14)71 (15)54 (14)0.57*1.82*Internet79 (13)70 (17)55 (18)0.54*1.29*0.000.740.58Family activitiesPaper83 (16)79 (18)70 (20)0.260.67*Internet81 (16)78 (17)66 (15)0.170.97*0.000.030.51Family cohesionPaper77 (21)68 (23)53 (26)0.380.89*Internet74 (20)68 (24)64 (28)0.240.380.000.180.05a The CHQ-CF scales ‘Role functioning-emotional–and ‘Role functioning-behavioral–were merged into a single scale in this studybDifference of the means divided by SD in the subgroup with chronic condition(s), where * indicates at least a “minimally important difference–(d ≥0.50) [28, 29]Role funct.-Emo/beh - Role functioning-emotional behavioral; Role funct.-Physical - Role functioning-physical
Discussion and conclusions
In this study we applied a randomized design to compare the results of the Child Health Questionnaire-Child Form (CHQ-CF) administered by a paper questionnaire and by an online questionnaire. The results provided support for the feasibility, internal consistency reliability, and construct validity of the CHQ-CF scales. Both modes of questionnaire administration yielded comparable scale scores and showed comparable psychometric properties. Additionally, the study provided reference/norm scores for clinical studies (general population of 13–7 year olds).
Strengths of the current study
The participation rate was high. Study group characteristics (age, gender, country of birth, and educational levels) were representative of those of the general population of Dutch adolescents [34]. Randomization to either the paper or internet mode of administration of the CHQ-CF was successful with respect to the evaluated characteristics.
Limitations
We applied a randomized parallel group design that allows for the comparison of indicators of feasibility, reliability, and validity at the group level between a subgroup that completed a paper version and a subgroup that completed an internet version of the CHQ-CF. However, this did not allow an evaluation of whether the same person would provide equivalent or different answers to the same CHQ-CF questionnaire administered by the alternative mode, which would require a randomized crossover design [25, 35]. Such an evaluation at the individual level requires the respondent to forget all previously provided answers at the second assessment, e.g., by waiting 1 or 2 weeks between the two measurements. It also requires that there is no effect from having previously completed a CHQ-CF questionnaire by any mode, paper or internet, at the second assessment, and that scores by the same mode of administration after a relatively short interval, in the absence of changes in health status, are exactly the same. However, in an evaluation of retesting with the same paper version of the CHQ-CF after 2 weeks, 5 out of 10 CHQ-CF scales showed statistically significant, higher scores at the second measurement with Cohen’s effect sizes ranging from 0.25 to 0.40, while intraclass correlation coefficients between the first and second measurement ranged from 0.06 thru 0.84 [7]. Furthermore, in a randomized crossover design, “carry-over–effects may be present, i.e., completing an internet version before a paper version may have a different effect on the second assessment, than does completing a paper version before an internet version [35]. Despite the logistical and the above-mentioned methodological challenges, we recommend future studies comparing the paper and internet versions of the CHQ-CF applying a randomized crossover design to evaluate congruency of answers to CHQ-CF items at the individual level.
In this study, internet and paper questionnaires were completed in a controlled environment with adequate privacy and supervision. This may not be the case during future applications. We are unaware of the impact less privacy during completion of the questionnaires may have, but this would apply to both the paper and the internet versions of the questionnaire.
For both modes of questionnaire administration, we did not evaluate correlations between CHQ-CF scores and a relevant parent-rated questionnaire such as the CHQ-PF50 [2, 3]. Test-retest reliability of the CHQ-CF and its responsiveness and sensitivity to changes in health were not evaluated in the current study. The CHQ-CF has a relatively large number of items; therefore, we recommend developing a shorter version in the future.
Psychometric properties
The psychometric properties, with only a few exceptions, were equal between the two modes of questionnaire administration. The Cronbach’s α of the scale “physical functioning–in the subgroup administered the paper version of the questionnaire was just under 0.70, and the difference with the alpha in the subgroup administered the internet version was not statistically significant.
Missing values
Compared with the paper version, the internet version was successful in reducing the quantity of missing data. Theoretically, differences in selective partial non-response between formats might have contributed to differences in scores that were reported in this study. In our study, in the subgroup (n = 86) that had at least one missing answer to a paper CHQ-CF item, all scale means were somewhat lower than in the subgroup (n = 389) with no missing answers, but these differences were not significant (P ≥0.05). Thus, missing answers are not a source of the observed score differences.
Score differences between modes of questionnaire administration
Recently, Ritter et al. found no statistically significant score differences between internet and paper modes of administration for 16 health-related measures, but the study was conducted in an opportunity sample retrieved from the internet, which limits its generalizability [23]. In a randomized internet-paper comparison among adolescents concerning various health measures other than the CHQ-CF, only one statistically significant score difference was reported among 21 topics [24]. In another randomized adolescent study, a medical consumption index and 11 indicators of fruit consumption and determinants of fruit consumption did not show statistically significant score differences between internet and paper administration of the questionnaire, except for one measure that showed small score differences between modes of administration [25]. The International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire did not show statistically significant score differences between internet and paper administration in two randomized adolescent studies [25, 26].
In our study, in the whole sample, the paper version resulted in slightly, yet statistically significant, higher scores for 4 of 10 CHQ-CF scales compared with the internet version. One plausible explanation is chance, since it should be considered that given multiple comparisons, there is a 1-in-20 chance of a false association for each comparison (Type I error at α = 0.05) [36]. A commonly used Bonferroni correction for 10 comparisons would imply an adjusted α =  0.05/10 =  0.005 [36]; at α =  0.005, only one score difference (regarding the scale “general behavior– was significant. Furthermore, given Cohen’s suggested guidelines for the interpretation of effect sizes, three of the four statistically significant differences between modes of administration can be considered as negligible (d ≤0.12), and one difference regarding the CHQ-scale “general behavior–(d = 0.21) can be considered as small [28]; all effect sizes were far below d = 0.50 that was suggested as an approximate threshold for “minimally important differences–by Norman et al. [29]. This study provides no explanations for the established small score differences between paper and internet administration, or for the established statistically significant, but small interaction effects of administration mode with age in the case of four CHQ-CF scales.
Conclusions
With increasing application of online health questionnaires rather than questionnaires on paper, especially in adolescent populations, it should be noted that comparison of results requires that the scores between these modes of administration do not show meaningful statistically significant differences. This study showed that, overall, paper and internet versions of the CHQ-CF yielded only a few, negligible or small, differences. Paper and internet modes of CHQ-CF administration may be combined in a single study, although researchers should consider the possibility of minor score differences depending on the mode of administration for some scales. We recommend repeated studies in other populations, including clinical populations, to confirm or reject our results.

child health questionnaire child form 87 items (chq-cf87)


Defense-Inducing Volatiles: In Search of the Active Motif

Herbivore-induced volatile organic compounds (VOCs) are widely appreciated as an indirect defense mechanism since carnivorous arthropods use VOCs as cues for host localization and then attack herbivores. Another function of VOCs is plant–plant signaling. That VOCs elicit defensive responses in neighboring plants has been reported from various species, and different compounds have been found to be active. In order to search for a structural motif that characterizes active VOCs, we used lima bean (Phaseolus lunatus), which responds to VOCs released from damaged plants with an increased secretion of extrafloral nectar (EFN). We exposed lima bean to (Z)-3-hexenyl acetate, a substance naturally released from damaged lima bean and known to induce EFN secretion, and to several structurally related compounds. (E)-3-hexenyl acetate, (E)-2-hexenyl acetate, 5-hexenyl acetate, (Z)-3-hexenylisovalerate, and (Z)-3-hexenylbutyrate all elicited significant increases in EFN secretion, demonstrating that neither the (Z)-configuration nor the position of the double-bond nor the size of the acid moiety are critical for the EFN-inducing effect. Our result is not consistent with previous concepts that postulate reactive electrophile species (Michael-acceptor-systems) for defense-induction in Arabidopsis. Instead, we postulate that physicochemical processes, including interactions with odorant binding proteins and resulting in changes in transmembrane potentials, can underlie VOCs-mediated signaling processes.
Introduction
Plants respond to herbivore damage with the release of volatile organic compounds (VOCs) that signal the presence of herbivore prey to predators and parasites and thereby serve as an indirect defense mechanism (e.g., Heil 2008). Research has demonstrated that these VOCs can also be perceived by neighboring plants or intact, systemic parts of the damaged plant (Baldwin et al. 2006; Heil 2008). Particularly, green-leaf volatiles (GLVs) have been associated with induced resistance in intact plants (Arimura et al. 2000; Engelberth et al. 2004; Farag et al. 2005; Ruther and Kleier 2005; Kost and Heil 2006;). However, little is known about the identity of VOCs that are active in this context or about a structural motif that active VOCs might have in common. In an attempt to explain the inducing activity of such VOCs on gene expression, it has been suggested that GLVs with an α,β-unsaturated carbonyl group such as (E)-2-hexenal can trigger defense responses in Arabidopsis through their activity as reactive electrophile species (Almeras et al. 2003).
In the present study, we used lima bean (Phaseolus lunatus L.) to search for traits that characterize defense-inducing VOCs. Lima bean responds to herbivore damage with the jasmonate-mediated production of VOCs and extrafloral nectar (EFN), an aqueous, sugar-containing secretion on nonreproductive plant organs that attracts predatory arthropods (mainly ants). The natural blend of VOCs that is released from a herbivore-damaged lima bean induces EFN secretion in intact neighboring plants (Kost and Heil 2006) and serves as a within-plant signal (Heil and Silva Bueno 2007). Among the quantitatively dominant VOCs that are released from induced lima bean, however, only (Z)-3-hexenyl acetate significantly induced EFN secretion when used as pure compound (Kost and Heil 2006). In the present study, we applied different structurally related esters to lima bean plants and monitored their EFN secretion.
Materials and Methods
Plants were cultivated from seeds collected in the coastal area of Puerto Escondido, Oaxaca, México (15°55.596 N and 097°09.118 W, elevation 15 m). Seedlings were cultivated under ambient conditions in 250 ml pots filled with soil from the original growing site. The plants were watered daily and fertilized 6 weeks after germination with commercial fertilizer: 10 ml per plant of a solution of 3 mg l−1 of “Fertilisante foliar de alta concentración” (Grupo Bioquímico Mexicano, Aaltillo, Coah., Mexico). Experiments were conducted with plants of an age of 8–10 weeks. The following compounds were dissolved in Lanolin paste (all at 0.1 μg μl−1): (A) (Z)-3-hexenyl acetate, (B) (E)-3-hexenyl acetate, (C) (E)-2-hexenyl acetate, (D) 5-hexenyl acetate, (E) (Z)-3-hexenyl isovalerate, (F) (Z)-3-hexenyl butyrate, (G) 2-ethylhexanol, (H) (E)-2-hexanal, (I) decanal, and (K) nonanal (Lanolin paste and all compounds were purchased from Sigma Aldrich). Each 0.25 g paste per plant was applied on green plastic stripes (MAX Bindeband) attached to the plant in order to avoid direct contact of the paste with the plant. The plants were then packed in perforated PET foil bags (Bratenschlauch, Toppits, Minden, Germany) and in nets to protect them from EFN consumers. Amounts of VOCs released from the lanolin paste into the plants’ headspace under these conditions were monitored in a parallel experiment that used a closed-loop stripping system as described previously (Kost and Heil 2006). EFN secretion was quantified from the five youngest leaves 24 h later. In short, EFN concentration was measured with a portable refractometer, and nectar volume was measured with glass capillaries (Kost and Heil 2006) to calculate the total amount of soluble solids secreted, which in the case of lima bean EFN are mainly glucose, fructose, and sucrose. Leaves were then collected and dried to calculate EFN secretion as soluble solids secreted per gram leaf dry mass and per 24 h. Plants to which Lanolin paste without any compound added had been applied served as controls (Lanolin control, LC). Per day, two groups each comprising all 11 treatments were investigated, and in total eight plants per treatment were used.
Results and Discussion
Amounts of VOCs released from the lanolin paste into the plant’s headspace resembled those released from an induced plant, i.e., all individual VOCs were present at 80–130% of the amount of (Z)-3-hexenyl acetate that is released under comparable conditions from five induced leaves of lima bean (data not shown, for amounts of VOCs released from an induced plant see Kost and Heil 2006). The different VOCs to which lima bean plants had been exposed affected their EFN secretion significantly (General linear model: F77,10 = 10.05, P < 0.001). Compounds A–F consisted of acyl hexenols in which the configuration and position of the double bond was systematically shifted from the polar head to the aliphatic terminus of the molecule. Moreover, the importance of the size of the acyl moiety was evaluated by using acetates, butyrate, and isovalerate of (Z)-3-hexenol. Although only one of the tested VOCs is naturally released from lima bean, exposure to the six compounds (A–F) elicited EFN secretions that were significantly higher than those of the controls (LSD posthoc analysis: P < 0.05, see Fig. 1). These compounds were (Z)-3-hexenyl acetate, (E)-3-hexenyl acetate, (E)-2-hexenyl acetate, 5-hexenyl acetate, (Z)-3-hexenyl isovalerate, and (Z)-3-hexenyl butyrate. Hence, EFN secretion by lima bean can be induced by VOCs that are not released naturally from this species, while several VOCs that are released from induced plants did not significantly change EFN secretion in previous experiments (Kost and Heil 2006).
Fig. 1EFN secretion in μg soluble solids secreted per gram leaf dry mass and per 24 h is depicted. Plants were exposed for 24 h to (A) (Z)-3-hexenyl acetate, (B) (E)-3-hexenyl acetate, (C) (E)-2-hexenyl acetate, (D) 5-hexenyl acetate, (E) (Z)-3-hexenyl isovalerate, (F) (Z)-3-hexenyl butyrate, (G) 2-ethylhexanol, (H) (E)-2-hexanal, (I) decanal, and (K) nonanal dissolved in Lanolin paste. Plants to which pure Lanolin paste had been applied served as controls (LC). Lower and upper whiskers represent the 5% and the 95% percentile, lower and upper margins of boxes the 25% and the 75% percentile, the lines within boxes indicate medians. Different letters appearing above boxes mark treatment effects that differ significantly from each other (P < 0.05 according to LSD posthoc analysis), and structures of compounds leading to a significant induction of EFN secretion as compared to the control are graphically presented. LogP values of all compounds were calculated with ChemDraw Ultra 6.0 and are given under the structures (compounds A–F) and in the insert (G–K)
How are VOCs perceived by plants, and via which mechanisms do they affect defense expression patterns? It has been suggested that molecules with an α,β-unsaturated carbonyl group can trigger defenses in Arabidopsis through their activity as reactive electrophile species (Almeras et al. 2003). In principle, VOCs could also be perceived by binding to specific receptor-proteins or to odorant binding proteins with a preference for a class of compounds, similar to animal olfactory systems.
Apparently, neither the configuration nor the position of the double bound is a critical factor. Compounds reported to prime or induce gene activity or phenotypic defenses in intact corn plants comprise (Z)-3-hexen-1-ol, (Z)-3-hexenal, and (Z)-3-hexenyl acetate (Engelberth et al. 2004; Farag et al. 2005; Ruther and Kleier 2005). We found that (Z)-3-hexenyl acetate, (E)-3-hexenyl acetate, (E)-2-hexenyl acetate, and 5-hexenyl acetate all elicited particularly high EFN secretion rates in lima bean. The majority of these substances lack an α,β-unsaturated carbonyl group, which thus cannot be a generally required motif. In addition, a compound such as 5-hexenyl acetate cannot yield an electrophile such as (E)-2-hexenal via the sequence of ester hydrolysis, oxidation, and isomerization, as it is principally possible for (E or Z)-3-hexenyl acetate or (E or Z)-2-hexenyl acetate. Moreover, unlike (Z)-3-hexenyl acetate the potential end product of the transformation sequence, (E)-2-hexenal, had only a weak effect on the nectar flow. Similarly, the observation that (Z)-3-hexenyl acetate and (E)-3-hexenyl acetate elicited almost identical EFN secretion rates contradicts the involvement of classical receptor proteins, since these usually require a specific stereochemistry of the interacting molecule. We further calculated LogP values to check for putative importance of the compound’s lipophilicity but did not find any clear relation between the EFN-inducing activity of a compound and its octanol-water partition coefficient (Fig. 1).
More studies are required to understand which VOCs induce plant defenses via which mechanism. However, our results point to a new mechanism. Changes in transmembrane potentials—occurring through modulations of ion fluxes—are involved in early signaling events in the cellular response to stress (Maffei et al. 2007), and exposition to VOCs indeed changes membrane potentials in intact lima bean leaves (M. Maffei, personal communication). It is thus tempting to speculate that the dissolving of VOCs in the membranes coupled to interactions with membrane proteins, similar to the odorant binding proteins of insects (Campanacci et al. 2001), leads to changes in transmembrane potentials and thereby induces gene activity. This hypothesis gains support from the observation that the induction of EFN secretion by VOCs appears to be a gradual one rather than a clear ‘yes-or-know’ response. Several of the compounds that are released naturally from lima bean showed a trend towards an increase in EFN secretion, although the difference was not significant: EFN secretion in response to Linalool was on average higher by 40% than in control plants, and DMNT ((3E)-4,8-dimethylnona-1,3,7-triene) increased EFN secretion by 50% (see Table 1 in Kost and Heil 2006). Similarly, plants exposed to ethylhexanol or (E)-2-hexanal in the present study showed a trend towards higher EFN secretions than controls (Fig. 1). EFN responds to a comparably wide variety of structures, and slight changes in the molecular structure gradually alter the induction effect. This observation is best explained by a comparably simple physicochemical process, whose detailed nature remains to be elucidated.
How can signals evolve that apparently lack chemical specificity? The answer might be simply that plants seldom are exposed to GLVs or chemically related compounds that are not released from attacked plants. In evolutionary terms, the probability of this situation was probably too low to cause any selection towards a more specific signal perception.

herbivore-induced volatiles
indirect defense
signal
hexenyl acetate
induced defense
plant-plant communication