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Received 9 October 1997; accepted after revision 1 May 1998.
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ABSTRACT |
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INTRODUCTION |
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The trigeminal mesencephalic nucleus (MNV) is a slender line of large pseudomonopolar neurones located on the border of the motor root of the fifth trigeminal nerve and the locus coeruleus in the brainstem (Pellegrino et al. 1979). The MNV contains the cell bodies of sensory primary afferent neurones that originate in the periodontal and jaw ligaments and relay proprioceptive sensations (at least in part from teeth), and may be important in jaw reflexes (Linden et al. 1994; Hassanali, 1997). Primary afferent MNV neurones are functionally similar to other proprioceptors in the body but unlike those neurones, which are encapsulated and located in discrete ganglia (for example the dorsal root and trigeminal ganglia), the MNV neurones are non-encapsulated and are retained within the brainstem (Liem et al. 1991; Linden et al. 1994; Terashima, 1996). In view of the central location of these neurones, some studies have examined the possibility that MNV neurones may receive synaptic contacts. However, electrophysiological studies have failed to identify synaptic currents in MNV neurones despite the fact that electron microscopy studies show that MNV neurones do receive a sparse synaptic innervation to their somata (Liem et al. 1991). On the basis of such data it seems likely that MNV primary afferent terminals in the periphery (jaw ligaments and periodontal layers) and their central terminals (in the trigeminal mesencephalic motor nucleus, the supratrigeminal nucleus, trigeminal principal sensory nucleus and part of the spinal trigeminal subnucleus oralis) are important in the detection, relaying and central processing of proprioceptive information (Lou et al. 1995; Dessem et al. 1997).
Electrophysiological recordings of the somatic properties of MNV neurones indicate that they display a marked membrane potential sag back towards resting membrane potentials during membrane hyperpolarization, a property termed anomalous inward rectification (Henderson et al. 1982). Similar anomalous inward rectification has been described in many central and peripheral neurone types (see Pape, 1996, for review), and in other sensory neurones, e.g. dorsal root ganglion neurones (Mayer & Westbrook, 1983), nodose and trigeminal ganglion neurones (Ingram & Williams, 1994, 1996; Jafri & Weinreich, 1998), the current underlying this rectification has been studied in detail using voltage-clamp recording. A voltage-clamp study of the currents that are activated by membrane hyperpolarization of MNV neurones is lacking, and thus the identity of the channels that underlie anomalous inward rectification in these neurones is unknown.
In the course of a previous study we directly measured the change in membrane conductance that occurred as a result of the P2X receptor-mediated inward current in MNV neurones by applying hyperpolarizing membrane potential steps (Khakh et al. 1997). We observed that hyperpolarizing the cells activated a voltage-dependent current that could underlie anomalous inward rectification. In the present study we have investigated this observation. Our first goal was to characterize hyperpolarization-activated currents in MNV neurones and then to relate the properties of these to similar currents already reported in the literature.
Preliminary reports of some of these findings have been presented (Khakh & Henderson, 1997a, b).
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METHODS |
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Preparation of brain slices
Preparation of pontine brain slices containing the MNV has been described previously (Henderson et al. 1982). Briefly, Wistar rats (postnatal days 14-28) of either sex were humanely killed by stunning and cervical dislocation. The scalp and skull were removed and the brain excised. A block of tissue containing the pons and cerebellum was mounted rostral surface uppermost on a plastic slide using cyanoacrylate glue. Serial slices were cut in gassed (95 % O2-5 % CO2) artificial cerebrospinal fluid (ACSF) solution comprising (mM): NaCl, 126; KCl, 2·5; NaH2PO4, 1·24; MgCl2, 1·3; CaCl2, 2·4; NaHCO3, 26; and D-glucose, 10, at 4°C using a Vibratome (Oxford, USA). Slices were cut at a setting of 200-230 µm, although actual slice thickness was not determined. Cut slices were maintained at ambient temperature (22-25°C) in gassed ACSF, and when required were transferred to the recording chamber (volume of chamber, 0·5 ml) mounted on a Zeiss Axioskop microscope and continually superfused (2·5 ml min-1) with gassed ACSF at 30°C. All slices were used within 6 h of dissection.
Electrophysiological recording
Cells were viewed using Nomarski optics and MNV neurones were identified as large diameter (
40 µm) cells lying lateral to the locus coeruleus (Henderson et al. 1982). Whole-cell recordings were made using fire-polished borosilicate glass electrodes of 2-4 M
resistance. The pipette-filling solution was of the following composition (mM); CsCl or KCl, 130, as indicated in the text; EGTA, 5; Hepes, 10; CaCl2, 1; MgCl2, 2; Na2GTP, 0·5; and MgATP, 5. Using these solutions there was a tip potential of -2 mV which has been accounted for in the results presented. Whole-cell recordings were made using the 'blow and seal' method on identified neurones. Visually we observed large and small neurones in the region of the MNV (Liem et al. 1991), with membrane capacitance of between 30 and 100 pF. In this study, neurones with membrane capacitance of between 40 and 80 pF were studied, since cells of capacitance less than 40 pF did not exhibit I h and cells of capacitance greater than 80 pF could not be reliably voltage clamped. Series resistance values were between 3 and 5 M and data are presented only from recordings where this did not change by more than 15 % over the period of recording. All voltage-step protocols were applied using pCLAMP 6.0 (Axon). Data were filtered at 2 kHz using an Axopatch 200B amplifier (Axon), displayed on a chart recorder (Gould, UK), and saved to digital audio tape (Biologic, UK) and computer for later analysis (digitized at 4-6 kHz).
All drugs were kept frozen (-20°C) as stock solutions until required and then diluted to the appropriate final concentrations and applied in the ACSF superfusing the slice. Drugs would reach the recording chamber after a period of 30-60 s after switching, and complete exchange of the bath fluid took less than 1 min. 4-(N -Ethyl-N -phenylamino)-1,2-dimethyl-6-(methylamino) pyridinium chloride (ZD7288) was a gift from Zeneca (UK).
All values reported are means ± standard error of the mean from the number of cells (n) as indicated in the text. Statistical significance was determined using Student's paired or unpaired t test, as appropriate.
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RESULTS |
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Initial observations
Membrane potential and membrane capacitance measured in 151 neurones were -63 ± 1 mV and 69 ± 1 pF, respectively. In initial studies we observed that hyperpolarizing MNV neurones by 10-50 mV (from -62 mV) activated a slow inward current in all neurones with capacitance > 40 pF.
In order to study the effects of membrane hyperpolarization on the membrane potential of MNV neurones, we performed initial experiments under current-clamp recording. Injection of hyperpolarizing current of between 1 and 2 nA caused MNV neurones to hyperpolarize. This hyperpolarization was not sustained and decayed during the pulse (Fig. 1). This phenomenon has previously been referred to as time-dependent anomalous inward rectification (Henderson et al. 1982). Application of Cs+ (1 mM) in the extracellular solution relieved this sag, such that for the same injection of hyperpolarizing current there was now a larger and sustained membrane hyperpolarization (Fig. 1). This was quantified as an increase in the integral area of the hyperpolarizing step from 12498 ± 1617 to 19110 ± 2381 mV ms-1 (P < 0·05; n = 4). In these same neurones, Cs+ (1 mM), which can block anomalous inward rectification (see above), did not change the resting membrane potential of MNV neurones (0·6 ± 0·5 mV change in resting membrane potentials on addition of Cs+; P > 0·05; n = 4). Furthermore, Cs+ (1 mM) did not affect the amplitude of the after-hyperpolarization (AHP) following single action potentials in MNV neurones. Action potentials were evoked by depolarizing neurones to threshold and AHPs measured as the difference between the resting membrane potential and the peak of the AHP; the AHP changed from -20·9 ± 0·95 mV in control to -19·1 ± 0·87 mV in Cs+ (P > 0·05; n = 4).
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The injection of hyperpolarizing current (top) caused a change in the membrane potential (bottom) which was followed by a sag in the membrane voltage response (indicated by arrowhead in the left trace). This time-dependent anomalous rectification was blocked by the extracellular application of Cs+ (1 mM; right trace). | ||
Biophysical properties
Anomalous inward rectification has been described in dorsal root ganglion neurones where the hyperpolarization-activated cationic current (Ih) underlies this phenomenon (Mayer & Westbrook, 1983). Therefore, we sought to investigate whether or not MNV neurones express Ih, which is the neuronal equivalent of the cardiac current termed If (Pape, 1996).
Application of step hyperpolarizations from a holding potential of -62 mV evoked membrane currents that consisted of two components (Fig. 2, top trace). The first was an instantaneous change in membrane conductance produced by stepping the cell to hyperpolarized potentials. The second was a slowly developing inward current which had characteristics that closely resembled those of Ih described previously in a number of neuronal preparations (Pape, 1996). A tail current was also present on returning the cell to -62 mV (Fig. 2, top trace). The latency between membrane hyperpolarization and the onset of the inward current was voltage dependent, being 14 ± 2 ms for a step to -92 mV, and 5 ± 1 ms for a step from -62 to -132 mV (n = 4). The size of the conductance change at the beginning of the voltage step was always smaller than that at the end of the step, indicating an increase in membrane conductance during the slow inward current. Application of extracellular Cs+ (1 mM) blocked all the current activated by hyperpolarization (Fig. 2, middle trace), and the pure hyperpolarization-activated current was revealed by digital subtraction of traces in the presence of Cs+ from those in control conditions (an example is shown in Fig. 2, bottom trace). The slow inward current did not inactivate during the hyperpolarization for steps up to 5 s duration. To examine the kinetics of the current, voltage steps of more than 2 s duration were used to ensure that the current was fully activated and that enough data were collected to allow analysis using exponential fitting functions (using the pCLAMP 6 Clampfit program). Activation kinetics were analysed using double-exponential functions because single exponentials produced unreliable fits. For a step from -62 to -132 mV the time constant for the initial phase of the slow inward current (t1) was 63 ± 5 ms and for the second phase (t2) was 2015 ± 816 ms (n = 5). Similarly, current deactivation examined by stepping back from a hyperpolarized potential could also be best described by double exponentials. For a step from -132 to -72 mV, t1 and t2 were 53 ± 5 and 307 ± 38 ms, respectively (n = 5).
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Hyperpolarizing steps from -62 to -132 mV evoked membrane responses in MNV neurones that consisted of three components. There was an initial change in instantaneous holding current, which was followed by a slowly developing inward current (shown as Ih activation in the top trace). On returning the membrane potential back to -62 mV, the tail current was measured as a residual current. The middle trace shows data from the same neurone in the presence of extracellular Cs+; note here there is no slow inward current. The bottom trace shows a digital subtraction of the middle trace from the top trace (Control - Cs+); the current that is revealed represents the pure hyperpolarization-activated current (see also Banks et al. 1993). | ||
To examine the voltage dependence of activation, tail currents were measured on return to -62 mV after stepping the cell to defined hyperpolarized potentials (Fig. 3). The hyperpolarization-activated current activated between -72 and -122 mV with a maximum Ih tail current amplitude (Imax) of -1261 ± 293 pA at -132 mV (n = 6). The activation curve could be fitted with a Boltzmann function with a V½ of -94 ± 3 mV and a slope value, k, of 8·4 ± 1·6 mV (Fig. 3C). The reversal potential of hyperpolarization-activated currents was estimated using previously described methods (Banks et al. 1993; Watts et al. 1996), and was found to be -57 ± 8 mV (n = 5).
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A, upper panel shows the voltage waveform used to activate Ih from a holding potential of -62 mV to defined potentials in 10 mV steps of 500 ms duration. Lower panel shows a family of Ih currents recorded from an MNV neurone. The data from this cell are plotted in B. The activation curve for the steady-state currents did not reach a maximum but that for the tail currents did. The data for tail currents from a number of cells (n = 6) are shown in C. In this graph the data from individual cells have been normalized to the size of the current at -132 mV using steps that are 2 s in duration, and the pooled data were fitted to the Boltzmann function of the form: I/Imax = [1 + exp ((Vm - V½)/k)]-1, where Vm is the membrane potential, V½ is the membrane potential at which Ih is half-activated, I is the tail current amplitude recorded after the voltage step back to -62 mV, Imax is the maximum tail current amplitude recorded after a step from -132 mV, and k is the slope factor that determines the steepness of the fitted curve. | ||
Pharmacological properties
We have used known blockers of I h and If to characterize pharmacologically the hyperpolarization-activated current in MNV neurones. Extracellular Cs+ has previously been shown to block I h (Pape, 1996), and in MNV neurones Cs+ rapidly and reversibly blocked the hyperpolarization-activated current (Fig. 4A -C) in a concentration-dependent manner with a pIC50 (log10 of the IC50) of -3·9 ± 0·2 and a Hill coefficient of 0·8 ± 0·3 (n = 4; Fig. 4C). In all cells tested, 1 mM Cs+ caused complete inhibition of the hyperpolarization-activated current, and this effect was rapid in onset (time to peak was < 1 min, which is equivalent to the solution exchange in the bath) and was rapidly reversible (time to 50 % recovery was 140 ± 42 s (n = 4), which is about 2-fold slower than bath washout; see Fig. 4B). The blocking effect of Cs+ occurred in a use-independent manner (n = 5; Fig. 4D). Extracellular Ba2+ (1 mM) did not affect the hyperpolarization-activated current when applied for periods of up to 5 min (n = 2; Fig. 4A).
In the heart, ZD7288 has been shown to inhibit If (BoSmith et al. 1993), and in the present study we sought to determine whether this compound could also affect the hyperpolarization-activated current in MNV neurones. ZD7288 (1 µM) produced a 70 ± 4 % inhibition of the hyperpolarization-activated current (n = 5). This effect was slow to develop, taking about 15 min to occur, and did not reverse with washout periods of up to 20 min (Fig. 4F). ZD7288 (1 µM) did not markedly change the holding current of MNV neurones (-2 ± 19 pA change; range, -43 to +30 pA; n = 4).
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A, extracellular Ba2+ (1 mM) had little effect on Ih, whereas in the same cell Cs+ (1 mM) caused complete block of Ih. B, the time course of action of Cs+ was rapid and was readily reversible. C, Cs+ blocked Ih in a concentration-dependent manner. In these experiments the data from a number of cells (n = 4) were normalized to the amplitude of Ih before Cs+ was added, and the resulting concentration-effect curve was fitted using iterative methods to the four-parameter logistic equation. D, Cs+ blocked Ih in a use-independent manner (n = 5). Ih was activated 5 times every 5 s ( | ||
Inhibition of hyperpolarization-activated currents by P2X receptor activation
MNV neurones express P2X receptors, which are ATP-gated cation channels (Khakh et al. 1997). During the course of our experiments we noticed that activating P2X receptor channels caused an inhibition of I h. We observed that applying ATP
S (30 µM) to activate P2X receptors in this nucleus evoked inward currents in all cells tested. These currents reached a peak and then desensitized over a time course of between 1 and 2 min (Fig. 5B). The amplitude of the I h tail current was measured using the following protocol. The cell was stepped from -62 to -132 mV to activate I h, and then stepped back to -62 mV to measure the I h tail current every 5 or 20 s. Over the duration of the experiment (usually 5 min) there was little run-down of I h (less than 5 % change; Fig. 5A). During the application of ATPS (30 µM) there was an inhibition of I h that closely matched the onset, activation, peak effect and desensitization of the ATPS-evoked inward current (Fig. 5B). ATPS (30 µM) evoked a peak inward current of -678 ± 176 pA, and the maximum I h inhibition was 84 ± 9 % in this population of MNV neurones (n = 7). Representative I h currents before, during and after activation of P2X receptors are shown in Fig. 5B and C.
We investigated whether calcium entry through P2X receptors plays a role in the inhibition of I h produced by P2X receptor activation. Recordings were made from six MNV neurones with the rapid calcium chelator BAPTA (11 mM) in the pipette solution, and compared with recordings from six MNV neurones with no BAPTA in the pipette solution (I h was activated by a step from -62 to -132 mV). The intracellular application of BAPTA did not change the properties of the current evoked by ATPS (30 µM; Fig. 5D, left-hand bar graph; n = 6). However, there was a significant diminution in the ability of ATPS to inhibit I h (Fig. 5D, right-hand bar graph; n = 6). The effect of P2X receptor activation on the voltage dependence of I h activation was investigated with and without 11 mM BAPTA in the pipette solution. In control cells with no BAPTA in the pipette solution, I h activation curves were determined before the application of ATPS (10 µM) and at the peak of the inward current resulting from ATPS application in order to determine whether activation of P2X receptors could change V½, k or Imax. P2X receptor activation with ATPS (10 µM) caused a significant 10 mV shift in the hyperpolarizing direction for the activation of I h in cells in which it had negligible effects on k and Imax (n = 4; Fig. 5E, right-hand graph). This suggests that activating P2X receptors elicited a change in the gating of Ih channels. In experiments with BAPTA in the pipette solution, application of ATPS (10 µM) failed to cause any significant change in the V½, k and Imax for I hactivation (P > 0·05; n = 6; Fig. 5E, right-hand graph).
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A, over a time course of up to 5 min there was no run-down of Ih (n = 4). B, holding current ( | ||
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DISCUSSION |
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We have identified the current activated by membrane hyperpolarization in MNV neurones as I h on the basis of its similarity to I h observed in other neuronal preparations (see Pape, 1996).
Pharmacologically, I h can be discriminated from other hyperpolarization-activated currents such as inward rectifier potassium currents because it is not blocked by intracellular Cs+ or extracellular Ba2+ (Womble & Moises, 1993). The use of the bradycardiac agent ZD7288, developed as an inhibitor of If (the cardiac hyperpolarization-activated cation current), provides further pharmacological evidence that I h is the neuronal equivalent of If. Indeed the degree and time course of inhibition of Ih by 1 µM ZD7288 in MNV neurones is almost identical to that seen for If (BoSmith et al. 1993). Ih was attenuated by extracellular Cs+ in a concentration-dependent manner with an IC50 value of 100 µM. The Hill coefficient of 0·8 calculated from the concentration-effect curve to Cs+, and the lack of use dependence suggest that Cs+ has only one binding site on I h channels and that it can bind without I h channels being open, or that it binds in the channel outer vestibule. At present it is unclear whether Cs+ and ZD7288 bind to the same site on I h channels, but they clearly differ in their kinetics of action. Under identical recording conditions, for a similar degree of block, ZD7288 was about 7 times slower than Cs+ in inhibiting I h.
The biophysical properties of latency to onset, activation, lack of inactivation, deactivation, voltage dependence and reversal potential of Ih described in the present study are generally similar to data reported for Ih in other neuronal preparations. Notably, Ih activation in MNV neurones could be described by two time constants, and this suggests that MNV neurones may express two populations of Ih channels with differing kinetics (see also Banks et al. 1993). Ih activation can differ markedly between different cell types, ranging from hundreds of milliseconds in some types of neurone to tens of seconds in others, and this may reflect the predominance of rapidly and slowly activating channels underlying Ih in differing cell types (Pape, 1996). However, direct comparison of biophysical properties such as V½ values to those published in other studies is problematic because of the different recording conditions used in each study, especially in view of the fact that temperature can have significant effects on Ih activation (Watts et al. 1996). However, on the basis of the described pharmacological and biophysical criteria we have provided evidence for the presence of I h in MNV neurones.
In the present study the lack of effect of Cs+ on the resting membrane potential or the evoked action potential AHP in MNV neurones suggests that I h does not contribute to the firing rate of MNV neurones. It will be important to investigate what role I h plays in the modulation of burst firing in neurones; these studies could not be performed in the present study because MNV neurones do not fire single action potentials or bursts of action potentials at rest (see Henderson et al. 1982). However, overall these findings are in accord with results from dorsal root ganglion neurones (Mayer & Westbrook, 1983), nodose ganglion neurones (Jafri & Weinreich, 1998), mid-brain dopaminergic neurones (Mercuri et al. 1995), neurones of the medial nucleus of the trapezoid body (Banks et al. 1993), dorsal lateral lemniscus nucleus neurones (Wen et al. 1997) and pituitary somatotrophs (Simasko & Sankaranarayanam, 1997), where I h does not contribute significantly to the resting and active properties of these neurones. However, in other neurones I h can contribute substantially to the resting and active properties of neurones, e.g. thalamic relay neurones (McCormick & Pape, 1990), hippocampal neurones (Maccaferri et al. 1993; Gasparini & DiFrancesco, 1997), neurones of the basolateral amygdala (Womble & Moises, 1993) and ventrobasal thalamic neurones (Williams et al. 1997). The reasons why I h is active at rest in some neurone types and not in others is unclear at present. One possibility is that basal modulation of I hin some neurones may shift its activation curve closer to resting membrane potential levels. An alternative explanation may be that there is heterogeneity in the channels that underlie I h (see Banks et al. 1993) and these may predominate in different neurone subsets. These and other issues may be best addressed when the gene that encodes the I h channel protein has been cloned and I h channels expressed and studied in isolation.
The possible contributions of I h to determining the resting membrane potential of neurones has been discussed (Pape, 1996), but the absence of such a role in MNV neurones may not preclude a physiological function. In MNV, dorsal root ganglion (Mayer & Westbrook, 1983) and primary auditory neurones (Chen, 1997), activation of I h during hyperpolarizing electrotonic potentials can cause a membrane potential overshoot that is Cs+ sensitive (see Fig. 1), which may decrease, or even reach the threshold for action potential firing. Furthermore, the physiological role for I h in sensory neurones is likely to be at the peripheral terminals and not at the soma (see Introduction). In accord, data have been presented to show that elevation of intracellular cAMP can shift the V½ of I hactivation to more depolarized levels closer to the resting membrane potential (Ingram & Williams, 1994), and I hhas been identified on sensory neurone growth cones at sites distant to the cell soma (Wang et al. 1997).
Numerous studies have shown that elevation of cAMP by transmitter receptors coupled to Gs proteins can shift the voltage dependence of I h in neurones and in cardiac myocytes (see Pape, 1996, for review). Recent data in cardiac myocytes and sensory neurones show that these actions may be the direct effects of cAMP on I h channels (DiFrancesco & Tortora, 1991; Ingram & Williams, 1996; Bois et al. 1997), and the overall contribution of protein kinases and the direct effect of cAMP on I h in many cell types remains unclear. In initial studies on MNV neurones we have demonstrated only a modest effect of cAMP elevation using forskolin, amounting to a 5 mV depolarizing shift in the V½ for I h activation (Khakh & Henderson, 1997a). In this respect the current identified as I h in MNV neurones appears intermediate in its sensitivity to cAMP. For example, I h in pituitary somatotrophs is not affected by cAMP elevation at all (Simasko & Sankaranarayanam, 1997), whereas most cell types are affected and this results in a V½ shift of between 5 and 20 mV (see Pape, 1996). In the present study we have reported initial findings indicating that activation of P2X receptor channels can cause an apparent inhibition of I h, which is associated with a hyperpolarizing shift in the V½ for activation. This P2X receptor-mediated inhibition of I h is blocked by intracellular BAPTA, suggesting that calcium entry through P2X receptors is likely to be involved in this effect. In cases where increases in intracellular cAMP can result in the modulation of I h, the result is always a depolarizing shift in the V½ for I h activation (Pape, 1996). Recent studies indicate that I h activation curves can also be shifted in the hyperpolarizing direction. In rat hypoglossal motoneurones, the action of clonidine acting at 
2 adrenoceptors and the action of substance P on NK1 receptors in nodose neurones causes a hyperpolarizing shift in the V½ for I h activation (Parkis & Berger, 1997; Jafri & Weinreich, 1998). The molecular mechanisms for both these effects, and that responsible for the inhibition of I h by P2X receptor activation reported in this study, are at present unknown. It is interesting to note, however, that both 2 adrenoceptors and NK1 receptors couple to G proteins, and their activation can result in the elevation of intracellular calcium levels (Aantaa et al. 1995; Khawaja & Rogers, 1996). P2X receptor channels are calcium permeable and would also be expected to increase intracellular calcium levels (Rogers & Dani, 1995; Rogers et al. 1997). In future work it will be interesting to determine the mechanism(s) by which I h can be modulated by calcium either released from intracellular stores or entering from the cell exterior, or possibly by a secondary calcium-dependent process. It will also be important to determine how these processes vary between different neurone types (see Budde et al. 1997; Luthi & McCormick, 1998).
I h channels exist on many cell types including brain and sensory neurones (see above, and also Pape, 1996). There is functional evidence for I h channels on growth cones (Wang et al. 1997) and axons (Grafe et al. 1997) of sensory neurones, providing strong evidence that I h may be located at primary afferent nerve terminals. It has been suggested that modulation of I h in primary afferent terminals of nociceptive neurones may contribute to hyperalgesia (Ingram & Williams, 1994, 1996). In this study we have provided the first evidence for the presence of I h in proprioceptive MNV neurones. It will be interesting to determine whether I h can contribute to the membrane properties of the central and peripheral terminals of proprioceptive neurones.
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Acknowledgements
This work was supported by GlaxoWellcome.
Corresponding author
B. S. Khakh: Division of Biology 156-29, California Institute of Technology, Pasadena, CA 911025, USA.
Email: balkhakh{at}cco.caltech.edu
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) from -62 to -132 mV, and then the slice was left to incubate in Cs+ (1 mM) for 5 min whilst the cell was clamped at -62 mV and Ih not activated. Subsequently, Ih was activated again, 5 times once every 5 s; note that Ih is maximally blocked at the first step (
). The slice was then placed in bathing medium that contained no Cs+ for 10 min whilst the cell was clamped at -62 mV. Subsequently, Ih was activated 5 times once every 5 s (
). E, ZD7288 (1 µM) caused ~70 % inhibition of Ih activated by steps from -62 to -132 mV. F, the blocking effect of ZD7288 was slow to develop and did not reverse with up to 17 min of washout. The residual current remaining in ZD7288 (1 µM) could be blocked by Cs+ (1 mM; data not shown).
) or without 11 mM BAPTA (
). However, the inhibition of Ih (right-hand bar graph) caused by activating P2X receptors was significantly reduced by dialysis with BAPTA (