| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Brain Research Institute, Medical University of Vienna, Vienna, Austria
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
(Received 26 August 2003;
accepted after revision 19 December 2003;
first published online 23 December 2003)
Corresponding author J. Sandkühler: Brain Research Institute, Department of Neurophysiology, Medical University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria. Email: juergen.sandkuehler{at}meduniwien.ac.at
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- and C-fibres, many of which are nociceptive (Willis & Coggeshall, 1991). In the rat, 5–10% of the lamina I neurones send projections to the brain (Bice & Beal, 1997a,b; Spike et al. 2003), thus relaying primary afferent information directly to nociceptive centres like the thalamus, the parabrachial area and the periaqueductal grey (PAG) (Marshall et al. 1996; Todd et al. 2000). There, discriminative evaluation of the painful stimuli takes place and autonomic and emotional responses and pain-related behaviour are generated (Bester et al. 2000; Gauriau & Bernard, 2002; Keay & Bandler, 2002). In addition, recent evidence indicates that lamina I projection neurones may have a decisive role in the development and maintenance of persisting pain. In the rat, the group of lamina I projection neurones seems to largely overlap the subset of lamina I neurones that express the NK1 receptor (Ding et al. 1995; Todd et al. 2000). Selective destruction of the NK1 receptor-expressing lamina I neurones by intrathecal application of the cytotoxin saporin conjugated to substance P strongly attenuates hyperalgesia, allodynia and central sensitization in rat models of inflammatory and neuropathic pain but leaves responses to acute, mildly painful stimuli unaffected (Mantyh et al. 1997; Nichols et al. 1999; Khasabov et al. 2002; Suzuki et al. 2002). Information on the properties of lamina I projection neurones is therefore important to understand how they code nociceptive information for transmission to the brain and how they contribute to central sensitization and persisting pain. Retrograde labelling from supraspinal targets allows identification of these neurones in a spinal slice preparation and selective recording of them with the patch-clamp technique (Ikeda et al. 2003). Using this approach, we have recently shown that in lamina I neurones with a projection to the parabrachial area or the PAG, conditioning stimulation of C-fibre input from the dorsal root induces synaptic long-term potentiation (Ikeda et al. 2003; and authors' unpublished observations), which is a cellular model of centrally mediated hyperalgesia (Sandkühler, 2000). C-fibre synapses with unidentified lamina I neurones were, in contrast, not potentiated. Here, we have analysed membrane and discharge properties and types of afferent input in spino-parabrachial and spino-PAG neurones in lamina I and compared them to the properties of unidentified neurones. |
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Stereotaxic injections were performed as previously described (Ikeda et al. 2003). Briefly, 18- to 23-day-old Sprague-Dawley rats were anaesthetized with a mixture of ketamine and xylazine (75 mg kg-1 and 7.5 mg kg-1I.P.) and placed in a stereotaxic apparatus. A small (< 1 cm) scalp cut was made and a hole was drilled into the skull bone over the targeted injection area. A 500 nl Hamilton syringe was used to inject 50–100 nl of DiI (1.25–2.5%) into the right lateral parabrachial area or into the right caudal and/or intermediate ventrolateral and lateral PAG. The head wound was closed with two stitches and inspected daily. No signs of infection were detected. After recovery from the anaesthesia, the animals fed and drank normally. No pain-related behaviour was observed in any of the animals. After a 2–4-day survival period, spinal cord slices were prepared as described below. The brain was removed, cooled to -20°C in isopentane and cryostat sections (30 µm thick) of the brainstem were obtained to allow histological verification of the injection site (Fig. 1A and B). Only recordings from animals where the injection site clearly involved either the parabrachial area or the PAG but not both were included in the present study.
|
The lumbar spinal cord was removed from 20- to 26-day-old Sprague-Dawley rats under deep ether anaesthesia. The rats were then killed by an overdose of ether. Transverse slices, 500 µm thick, most of them with an attached dorsal root (6–12 mm long), were cut on a microslicer (DTK-1000, Dosaka, Kyoto, Japan). Slices were stored in an incubation solution (mM: NaCl 95, KCl 1.8, KH2PO4 1.2, CaCl2 0.5, MgSO4 7, NaHCO3 26, glucose 15, sucrose 50, oxygenated with 95% O2, 5% CO2; pH 7.4, measured osmolality 310–320 mosmol kg-1). For recording, a single slice was transferred to the recording chamber where it was superfused by recording solution at 3 ml min-1 at room temperature (20–24°C). The recording solution was identical to the incubation solution except for (mM): NaCl 127, CaCl2 2.4, MgSO4 1.3 and sucrose 0. All procedures used conformed to the guidelines of the Austrian Federal Ministry for Education, Science and Culture.
Patch-clamp recording
Dorsal horn neurones were visualized with Dodt-infrared optics. Retrogradely labelled spino-parabrachial and spino-PAG neurones in lamina I detected by epifluorescence and unidentified (not labelled) lamina I neurones were recorded in the whole-cell patch-clamp configuration with glass pipettes (2–6 M
) filled with internal solution (mM: potassium gluconate 120, KCl 20, MgCl2 2, Na2ATP 2, NaGTP 0.5, Hepes 20, EGTA 0.5, pH 7.28 with KOH, measured osmolality 300 mosmol kg-1) as described elsewhere (Ruscheweyh & Sandkühler, 2002). Voltage- and current-clamp recordings were made using an Axopatch 200B amplifier and the pCLAMP 8 or 9 acquisition software (Axon Instruments). Signals were low-pass filtered at 2–5 kHz, amplified 5-fold, sampled at 5–10 kHz and analysed offline using pCLAMP 9. Series resistance was usually between 5 and 25 M. No correction for the liquid junction potential was made. The mediolateral location of the recorded lamina I neurone was assessed at the end of the experiment by visually inspecting the position of the pipette tip under a 4 x objective.
Passive membrane properties
The membrane potential measured immediately after establishing the whole-cell configuration was called the ‘resting membrane potential’ even though the membrane potential of a neurone in a slice preparation superfused by artificial cerebrospinal fluid and measured in the whole-cell configuration is probably not equivalent to the situation in the intact animal. Only neurones that had a resting membrane potential more negative than -50 mV were investigated further. Membrane resistance and capacitance were calculated from the reaction to 100 ms-long hyperpolarizing voltage steps from -70 to -80 mV. The responses to 20 such voltage steps were averaged and the membrane resistance was then calculated from the difference in steady-state current at the two voltages. The total membrane capacitance was calculated from the area under the capacitive transient, corresponding to the charge moved by the voltage step. The built-in pCLAMP membrane test was found to be rather unreliable in determining the membrane resistance, especially for the large projection neurones where the capacitive transient often decayed with several time constants. In general, membrane resistances estimated with the pCLAMP membrane test were too low by a factor of 2–3 even for the smaller unidentified lamina I neurones. This accounts for the difference between the currently (Table 2) and previously (Ruscheweyh & Sandkühler, 2002) reported membrane resistances of unidentified lamina I neurones. In contrast, in small, compact neurones or the pCLAMP model cell, both methods give the same results.
|
Firing patterns were determined in response to depolarizing (usually 25–350 pA in 25 pA steps) current injections of 1 s duration. Firing patterns were routinely elicited from different holding potentials (at least one from between -50 and -65 mV, one from between -65 and -80 mV and one from a potential more negative than -80 mV) to detect voltage dependence of the firing patterns. Voltage-clamp recordings from different holding potentials were used to investigate the underlying currents. The action potential threshold as reported here was measured by means of a voltage step protocol. Holding potential was -80 mV and increasing voltage injections (usually -60 to -30 mV at 2 mV steps, modified if the action potential threshold did not fall into this range) were used to determine the threshold of the fast Na+ current. This method has the advantage of taking into account the facilitating or inhibiting effect of other voltage-dependent membrane currents like the A- and T-currents reported here on the action potential generation.
The action potential width was determined at the base of the first action potential evoked by depolarizing current injected to determine the firing pattern from a holding potential around -80 mV. The afterdepolarization amplitude was determined from the same action potential, measuring from the point of maximal afterhyperpolarization to the point of maximal afterdepolarization.
Primary afferent stimulation
The dorsal root was stimulated through a suction electrode with a constant current stimulator (WPI, Sarasota) at 0.1 ms pulse width. Excitatory postsynaptic currents (EPSCs) were classified according to their latency and threshold to be A- or C-fibre-evoked as previously described (Chen & Sandkühler, 2000; Ruscheweyh & Sandkühler, 2002). Constant latencies and absence of failures during 10 Hz stimulation (for A-fibres) or 1 Hz stimulation (for C-fibres) were used as criteria for apparently monosynaptic transmission.
Intracellular labelling with Lucifer yellow
In a separate set of experiments, we included Lucifer yellow (0.1%) in the patch pipette solution and filled lamina I unidentified and projection neurones for about 10–20 min. This was not routinely done because we could not exclude the possibility that Lucifer yellow might alter the membrane properties of the recorded neurones (Higure et al. 2003). At the end of the recording, the patch pipette was carefully withdrawn from the recorded neurone and the slice was stored in 4% paraformaldehyde in phosphate buffer. For analysis, the slice was cleared in DMSO and inspected under a fluorescence microscope equipped with a CCD camera (Olympus DP50). Two-dimensional reconstructions of the filled neurones in the transverse plane were made using the analySIS Software (Olympus). The same software was used to measure the somatic area and the length of the dendritic tree (sum of all visible dendrites). The mediolateral and dorsoventral extent of the dendritic tree were measured according to the method of Lima and Coimbra (Lima & Coimbra, 1986; Galhardo & Lima, 1999). For neurones lying in the medial two thirds of lamina I, the mediolateral axis was defined to be parallel to the dorsal horn surface while for neurones lying in the lateral third of lamina I, the mediolateral axis was taken to be the tangent to the dorsal horn surface at the location of the neurone. The dorsoventral axis was perpendicular to the mediolateral axis. In some neurones, very long, uniformly thin, seldom-branching processes were seen that probably correspond to axons. These processes were not included in the measurements of the dendritic tree.
Statistical analysis
All values are means ±S.E.M. One-way ANOVA, the non-parametric Mann-Whitney rank-sum test, Student's unpaired t test, Fisher's exact test and the 
2 test were used for statistical comparisons where appropriate. ANOVA was followed by a Mann-Whitney test or a t test corrected by the Bonferroni adjustment.
Drugs
Drugs and their sources were as follows: 4-aminopyridine (4-AP, 0.5–5 mM), tetraethylammonium (TEA, 20–30 mM) and nickel cloride (Ni2+, 100 µM) were from Sigma (Deisenhofen, Germany), cadmium chloride (Cd2+, 200 µM) from Merck (Hohenbrunn, Germany), tetrodotoxin (TTX, 0.5 µM) from Tocris (Bristol, UK), Lucifer yellow CH dipotassium salt from Fluka (Buchs, Switzerland), and 1,1'-didodecyl-3,3,3',3',-tetramethylindocarbocyanine perchlorate (DiIC12(3), DiI, 1.25–2.5%) from Molecular Probes (Leiden, The Netherlands). Stock solutions were prepared by dissolving the drugs in acidic buffer (pH 4.8, tetrodotoxin) or distilled water (4-AP, Cd2+, Ni2+) and stored in aliquots at -20°C. Drugs were added to the superfusion solution at defined concentrations as indicated. DiI was dissolved in DMSO to its final concentration and stored at 4°C. Lucifer yellow was freshly dissolved in pipette solution to a concentration of 0.1% where indicated.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
|
Apart from the five firing patterns (tonic, delayed, initial bursting, single spike, phasic bursting) that we have previously described in unidentified lamina I neurones (Ruscheweyh & Sandkühler, 2002) and that are not illustrated here, two additional firing patterns were identified. The gap firing pattern showed a long first interspike interval, followed by tonic firing. For just suprathreshold current injections, a long delay to the generation of the first action potential occurred (Fig. 2A). This firing pattern could be evoked only from holding potentials more negative than about -75 mV (Fig. 9A and B). The bursting firing pattern was characterized by a short burst of two to four action potentials riding on a slow depolarizing wave at the onset of a depolarizing current pulse. With stronger current injections, it was followed by tonic firing (Fig. 2B). This firing pattern was evident only if the neurone was held at a potential more negative than about -60 mV (Fig. 10A). From less negative holding potentials, rebound action potentials were evoked by release from hyperpolarizing current (not shown). These two novel firing patterns were typical of projection neurones (Fig. 3). Seventy-five per cent of the 84 spino-parabrachial neurones showed the gap firing pattern. Most of the 82 spino-PAG neurones exhibited either the gap or the bursting firing pattern. Both were almost absent from the population of unidentified neurones that showed a distribution of firing patterns comparable to our earlier findings (Fig. 3 and Ruscheweyh & Sandkühler, 2002). Three of the spino-PAG neurones showed a bursting firing pattern from a holding potential of about -70 mV and a gap firing pattern from about -85 mV and were grouped among the bursting neurones. No correlation was found between the mediolateral location of the neurones and their firing pattern (not shown).
|
|
|
|
|
Delayed firing is the second to third most frequent pattern in unidentified lamina I neurones (Fig. 3 and Ruscheweyh & Sandkühler, 2002). It is similar to the gap firing pattern encountered in projection neurones in that both displayed a voltage-dependent delay in action potential firing in response to a depolarizing current injection. In delayed firing neurones, the delay occurred before the first action potential while in gap firing neurones the delay was reflected by a long first interspike interval (Fig. 5). A voltage-dependent, rapidly activating and inactivating potassium current called A-current is responsible for the delayed firing pattern (Ruscheweyh & Sandkühler, 2002). Voltage-clamp analysis showed that gap firing neurones also exhibited an A-current. It will be called slow A-current because it had noticeably slower kinetics than the fast A-current belonging to the delayed firing pattern. Comparison of the two A-currents showed significant differences in voltage dependence, kinetics and pharmacology. Voltage-dependent activation curves were similar but removal of steady-state inactivation required significantly more negative holding potentials in the slow A-current than in the fast A-current (Fig. 6). This fits with the observation that the gap firing pattern could only be evoked from holding potentials more negative than about -75 mV (Fig. 9A and B) while a potential more negative that -60 mV usually was sufficient for the delayed firing pattern (not shown, see also Ruscheweyh & Sandkühler, 2002). Superposition of the two currents illustrates the difference in kinetics (Fig. 7A). Both the time to peak and the time constant of decay were voltage-dependent and significantly slower in the slow A-current than in the fast A-current (Fig. 7A and B, n= 5, P < 0.01). Recovery from inactivation was also significantly slower in the slow A-current (43 ± 8 ms, n= 6) than in the fast A-current (14 ± 2 ms, n= 6, P < 0.01, Fig. 8). Sensitivity to 4-AP was different in the two currents. 4-AP at 5 mM completely blocked the slow A-current but only partially blocked the fast A-current (to 21 ± 7% of control, n= 5, measured at a voltage step from -100-0 mV, Fig. 6A and B). At a lower concentration (0.5 mM), 4-AP does not affect the fast A-current (Ruscheweyh & Sandkühler, 2002) but it reduced the slow A-current to approximately 50% of control (n= 4, not shown). Consistent with this pharmacology, 4-AP (2–5 mM) abolished the gap in gap firing neurones (n= 5, Fig. 9C).
|
|
|
|
The bursting firing pattern was voltage-dependent, converting into a tonic firing pattern, albeit often with a strong frequency adaptation, when the neurone was held at a potential more depolarized than about -60 mV (n= 10, Fig. 10A). Block of the action potentials with TTX (0.5 µM) in burst firing neurones revealed a transient depolarizing wave in response to the current injection (n= 11, Fig. 10B). The corresponding transient inward current could be seen in voltage-clamp recordings and activated well below the action potential threshold (-60 ± 2 mV, n= 4, not shown). This current was not affected by TTX but was partially blocked by Ni2+ (100 µM, n= 4, not shown, block to 
50% of control) and Cd2+ (200 µM, n= 2, not shown, block to 10 and 50% of control), suggesting that it was a Ca2+ current. We did not further investigate this presumably low-threshold Ca2+ current because we did not find a combination of Ca2+ channel antagonists that reliably blocked the Ca2+ currents activating at higher voltages without affecting the low-threshold current described here.
Active and passive membrane properties
Lamina I neurones with different brainstem projections showed prominent differences in the shape of action potentials and their afterpotentials. Unidentified neurones had either a monophasic (Fig. 11A) or a polyphasic afterhyperpolarization as previously described (Ruscheweyh & Sandkühler, 2002). Seventy-eight per cent of the 96 spino-parabrachial neurones tested exhibited a hump in the falling phase of the action potential (Fig. 11B), leading to an increased action potential width compared to the other groups of neurones (Table 2 and Ikeda et al. 2003). No correlation between the presence of a hump and a specific firing pattern was found among the spino-parabrachial neurones. Humps in the falling phase of the action potential were not seen in unidentified neurones. Nineteen per cent of the 83 spino-PAG neurones tested showed a hump but none of the 29 burst firing spino-PAG neurones did. Spino-PAG neurones, however, often showed a prominent afterdepolarization following an action potential (Fig. 11C). Afterdepolarizations in spino-parabrachial neurones and unidentified neurones were rare and, if present, much smaller in amplitude (Table 2).
|
When neurones were grouped not according to projection but according to firing patterns (results summarized in Table 3), it became evident that burst and gap firing neurones largely differ in their membrane properties. Most prominent was the high membrane excitability of burst firing neurones. Differences in excitability and other properties were also found between the gap and burst firing neurones and neurones exhibiting the tonic and delayed firing patterns that are the patterns most frequently encountered in unidentified neurones.
|
In 143 neurones, we tested if electrical stimulation of A- and/or C-fibres in the dorsal root evoked excitatory postsynaptic currents (EPSCs). This was significantly (P < 0.05) more often the case in projection neurones (81% of the tested spino-parabrachial neurones, 87% of the tested spino-PAG neurones) than in unidentified neurones (69% of the tested unidentified neurones). The results are summarized in Table 4. Most prominent was the fact that few of the unidentified neurones but one-third of the spino-parabrachial neurones and almost two-thirds of the spino-PAG neurones received monosynaptic input from primary afferent C-fibres (P < 0.01 for comparison of unidentified neurones with spino-PAG neurones). Spino-PAG neurones seldom received input from primary afferent A-fibres, and if they did, the EPSCs were significantly smaller in amplitude than those evoked by stimulation of C-fibres (P < 0.01, Table 4).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The spino-parabrachial and spino-PAG pathways are thought to be involved in autonomic and emotional responses to painful stimuli and in the ascending part of an antinociceptive feedback loop (Bester et al. 2000; Millan, 2002; Keay & Bandler, 2002). In contrast, the spino-thalamic pathway is supposed to subserve discriminative evaluation of painful stimuli (Gauriau & Bernard, 2002). However, most lamina I neurones projecting to the thalamus or to the PAG have collaterals to the parabrachial area (Hylden et al. 1989; Spike et al. 2003). Similarly, the populations of lamina I neurones projecting to the parabrachial area and the caudal ventrolateral medulla seem to largely overlap (Spike et al. 2003) and all these pathways to supraspinal targets share a similar proportion of NK1 receptor expressing neurones (Marshall et al. 1996; Todd et al. 2000). It is therefore tempting to hypothesize that a uniform population of lamina I nociceptive projection neurones sends its axon collaterals to several supraspinal targets where their information can be used either for discriminative or more integrative purposes. However, we found two very distinct groups of projection neurones: the gap firing and the burst firing neurones. Burst firing neurones had easily excitable membranes, narrow action potentials, pronounced afterdepolarizations and frequent monosynaptic input from primary afferent C-fibres and were found preferentially among the spino-PAG neurones. Gap firing neurones had less excitable membranes and broad action potentials due to a hump in the falling phase of the action potential. Gap firing neurones were preferentially encountered among the spino-parabrachial neurones but also in about half of the spino-PAG neurones. Burst firing neurones seemed to be a more specific input to the PAG. As most lamina I spino-PAG neurones have collaterals to the parabrachial area, they constitute a subgroup of about one-third of the spino-parabrachial population. Nevertheless, they have been shown to differ from other spino-parabrachial neurones, e.g. by exhibiting weaker immunoreactivity for the NK1 receptor than other lamina I projection neurones (Spike et al. 2003). Together with our finding that spino-PAG neurones exhibit firing patterns and membrane properties that set them apart from the average of spino-parabrachial neurones, it seems that the spino-PAG neurones may represent a distinct subclass of lamina I projection neurones. The functional significance of these two types of lamina I projection neurones remains to be elucidated. We have recently shown that they express synaptic long-term potentiation in response to different patterns of primary afferent stimulation (Ikeda et al. 2003 and authors' unpublished observations). Firing patterns may also be related to the physiological type of the neurone, as suggested by Han et al. (1998) for unidentified lamina I neurones. Nociceptive-specific, polymodal nociceptive and thermoreceptive-specific cells are all encountered among lamina I projection neurones in the cat (Craig et al. 2001). About 80% of the spino-parabrachial neurones in rat and cat are nociceptive-specific (Hylden et al. 1985; Light et al. 1993; Bester et al. 2000), and 75% of the spino-parabrachial neurones in the present study were of the gap firing type. In vivo studies will be needed to investigate if there is a correlation between firing pattern and responses to natural stimulation in lamina I projection neurones.
The PAG is functionally organized in longitudinal columns. Lamina I sends projections to both the lateral PAG that mediates flight behaviour in response to escapable painful stimuli and the ventrolateral PAG that mediates passive behaviour in response to inescapable painful stimuli (Keay & Bandler, 2002). Unexpectedly, we found that the proportion of bursting to gap firing spino-PAG neurones depended on the rostrocaudal position of the injection site. Some functional organization in the rostrocaudal axis has been proposed for the lateral PAG, where the caudal and intermediate portions seem to be responsible for flight and confrontational defence, respectively (Bandler & Shipley, 1994). We do not know if the firing patterns of spino-PAG neurones relate to these functional aspects.
In vivo single-cell recordings have shown lamina I spino-parabrachial neurones to be nociceptive, mostly nociceptive-specific in the rat (Bester et al. 2000) and the cat (Hylden et al. 1985; Light et al. 1993), and to receive convergent input from primary afferent A- and C-fibres in the rat (Bester et al. 2000). In any spinal slice preparation, it is likely that part of the primary afferent input is lost because of damage to some afferent fibres during the slicing process. We found convergent input from A- and C-fibres in 45% of the spino-parabrachial neurones that received input from the segmental dorsal root. Lamina I neurones with a projection to the midbrain (PAG and surrounding structures) have been reported to be mainly nociceptive-specific and to receive primary afferent input from A-fibres in 97% and C-fibres in 20%in vivo (Hylden et al. 1986). We studied neurones with a projection to the PAG and found input from C-fibres in 94% and input from A-fibres in 25% of the neurones receiving input from the segmental dorsal root. Most prominent, however, was the high incidence of monosynaptic input from primary afferent C-fibres to our population of projection neurones. This and their high membrane excitability might render them especially susceptible for sensitization by nociceptive stimuli.
Unidentified lamina I neurones were different from the projection neurones in almost every aspect investigated in the present study. Most of our unidentified neurones probably were propriospinal neurones. It has been estimated that only 5–10% of lamina I neurones have a supraspinal projection (Bice & Beal, 1997a; Bice & Beal, 1997b; Spike et al. 2003). The chance to record from a projection neurone without the aid of retrograde labelling is therefore low. In our sample, 8% of the unidentified neurones showed the gap or bursting firing pattern typical of projection neurones, suggesting that some projection neurones were indeed recorded by chance.
The distribution of firing patterns and membrane properties of unidentified lamina I neurones reported here compares mostly well to what we found previously (Ruscheweyh & Sandkühler, 2002). A study on unidentified lamina I neurones in parasagittal spinal cord slices from adult rats revealed similar firing patterns but reported a larger proportion of single spiking versus tonic firing neurones (Prescott & De Koninck, 2002). This could be due to the slicing technique, the slicing plane or the age of the animals. Lamina I neurones are known to have large rostrocaudal dendritic arbors (Lima & Coimbra, 1986) that may be damaged by the transverse slicing technique. This might cause a selection bias or modify membrane properties. However, the infrared video contrast microscopy used here allowed us to record from neurones as deep as 100 µm from the surface in 500 µm thick slices, making it quite unlikely that a large proportion of any particular type of neurone was selectively destroyed or damaged. On the other hand, in the study of Prescott & De Koninck (2002), Lucifer yellow was routinely included in the intracellular solution, and this may modify membrane properties (Higure et al. 2003). In support of this explanation, in our sample of Lucifer yellow-filled unidentified neurones, 55% of 11 neurones showed the single spike firing pattern, while this was the case only for 9% of the 65 neurones recorded without Lucifer yellow (P < 0.01). Overall resting membrane potentials were more negative in the present than in our previous study. We attribute that to an improved recording technique yielding better seals. Membrane resistances were much higher in the present study. This is due to the modified analysis of passive membrane properties as described in Methods.
Intracellular labelling
In rat and cat, lamina I neurones have been morphologically classified into the fusiform, multipolar, pyramidal and flattened subtypes (Lima & Coimbra, 1986; Galhardo & Lima, 1999). It has been reported that in the rat, lamina I spino-parabrachial neurones are mostly fusiform while spino-PAG neurones are mostly pyramidal (Lima & Coimbra, 1989). This view has recently been challenged by Spike et al. (2003) who found that in the rat, both spino-parabrachial and spino-PAG neurones are about evenly distributed among fusiform, pyramidal and multipolar (including flattened) neurones. Unequivocal morphological classification of lamina I neurones according to Lima and Coimbra (Lima & Coimbra, 1986; Galhardo & Lima, 1999) is not possible in the transverse plane. We have therefore abstained from classifying the neurones of the present study by their morphology. In our transverse spinal cord slice preparation, a majority of projection neurones had prominent mediolateral dendrites, thus probably belonging to the pyramidal or flattened classes. In contrast, many unidentified neurones had a more ventral orientation of their dendritic trees, probably corresponding to the multipolar or fusiform type. Similar to our results, in vivo intracellular labelling of lamina I spino-parabrachial neurones in the cat produced mainly neurones with dendrites confined to lamina I and a considerable mediolateral extent (Light et al. 1993). In many of our projection neurones, long axons were seen that coursed laterally, often reaching the lateral edge of the dorsal horn, similar to what has been reported in the cat (Light et al. 1993). Spino-parabrachial neurones send their ascending axons through the dorsolateral funiculus but often have collaterals in the dorsal horn (Hylden et al. 1989; Light et al. 1993). We do not know if the axons seen here were ascending or segmental collaterals.
Firing patterns and responses to natural stimulation of the skin have been reported to be related to the morphological type of lamina I neurones in the rat (Prescott & De Koninck, 2002) and the cat (Han et al. 1998), respectively. These results have been obtained in unidentified lamina I neurones. Further investigations will be needed to determine if these findings extend to projection neurones.
The gap and delayed firing patterns and the underlying A-currents
The gap firing pattern is not a common one in the nervous system but it has been described, e.g. in the superior colliculus (Saito & Isa, 2000) and in vagal motoneurones (Yarom et al. 1985). In olivocochlear neurones, both the delayed and the gap firing pattern occur. As reported here for the spinal dorsal horn, they belong to two groups of neurones with different functions and projections (Fujino et al. 1997). Similar to our results, in olivocochlear neurones the A-current responsible for the gap firing pattern had slower kinetics than the A-current belonging to the delayed firing pattern. Apparently, the slow activation of the A-current in gap firing neurones allows for just one action potential to occur before the gap at the beginning of a depolarizing current pulse. The D-current is another voltage-dependent K+ current with slow kinetics (Storm, 1988). However, it is highly sensitive to 4-AP and usually completely blocked by 30–100 µM, which was not the case for the slow A-current described here.
The mean resting membrane potentials of gap and delayed firing neurones (-63 and -68 mV, respectively) fall into the steep regions of their respective inactivation curves (Fig. 6C). Thus, small variations of the resting membrane potential may have large impacts on A-current-modulated features of the neurone, among them action potential width, firing frequency and firing patterns (Rogawski, 1985). Excitatory postsynaptic potentials (EPSPs), especially large ones, are shortened in neurones expressing A-currents, which may be important for temporal summation of synaptic inputs (Cassell & McLachlan, 1986). As a general rule, A-currents reduce the excitability of neurones (Banks et al. 1996), especially when the membrane is already hyperpolarized by other factors such as inhibitory synaptic input. One might call this a potentiation of inhibition and hypothesize that, in the presence of intact segmental and descending inhibition, this mechanism protects gap firing projection neurones from sensitization.
The bursting firing pattern
The bursting firing pattern has not previously been described in the spinal dorsal horn. However, rebound spikes in response to release from hyperpolarizing current may have a similar mechanism and have been reported in a few dorsal horn neurones (Jiang et al. 1995; Ruscheweyh & Sandkühler, 2002). In the brain, the bursting firing pattern is more common and has been attributed to low threshold voltage-dependent Ca2+ channels (T-current) (Destexhe et al. 1998), high threshold voltage-dependent Ca2+ channels (Jung et al. 2001) or TTX-sensitive persistent Na+ channels (Azouz et al. 1996). In the present study, the slow depolarizing wave on which the burst rides persisted in TTX (Fig. 10B). The underlying current seemed to be a Ca2+ current as it was partially blocked by Ni2+ and Cd2+. Even if full characterization of the current was not possible, its low activation threshold (-60 mV) and its inactivation at similar voltages, inferred from the conversion of the firing pattern from bursting to tonic at these voltages (Fig. 10A), suggest that it may be a T-current (McRory et al. 2001; Perez-Reyes, 2003).
Low-threshold Ca2+ channels mediate Ca2+ entry during excitatory postsynaptic potentials (EPSPs) and action potentials (Kozlov et al. 1999) and increase the neuronal excitability by ‘boosting’ EPSPs and thus lowering the threshold for action potential generation (Huguenard, 1996). They also increase the EPSP duration, facilitating temporal summation (Williams & Stuart, 1999). In addition, they are able to convert inhibitory signals into excitatory ones by generating postinhibitory rebound spikes, especially when the resting membrane potential is both near the threshold for activation and inactivation as in the present study (Destexhe et al. 1998).
In conclusion, we have shown that lamina I unidentified neurones, most of them probably propriospinal, have properties that are fundamentally different from those of lamina I neurones with a supraspinal projection. The projection neurones, however, are also not a homogeneous group. The spino-PAG neurones seem to constitute a small but distinctive subgroup of the lamina I projection neurones with respect to firing patterns and membrane properties.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Bandler R & Shipley MT (1994). Columnar organization in the midbrain periaqueductal gray: modules for emotional expression?Trends Neurosci 17, 379–389.[CrossRef][Medline]
Banks MI, Haberly LB & Jackson MB (1996). Layer-specific properties of the transient K+ current (IA) in piriform cortex. J Neurosci 16, 3862–3876.
Bester H, Chapman V, Besson JM & Bernard JF (2000). Physiological properties of the lamina I spinoparabrachial neurons in the rat. J Neurophysiol 83, 2239–2259.
Bice TN & Beal JA (1997a). Quantitative and neurogenic analysis of neurons with supraspinal projections in the superficial dorsal horn of the rat lumbar spinal cord. J Comp Neurol 388, 565–574.[CrossRef][Medline]
Bice TN & Beal JA (1997b). Quantitative and neurogenic analysis of the total population and subpopulations of neurons defined by axon projection in the superficial dorsal horn of the rat lumbar spinal cord. J Comp Neurol 388, 550–564.[CrossRef][Medline]
Cassell JF & McLachlan EM (1986). The effect of a transient outward current (IA) on synaptic potentials in sympathetic ganglion cells of the guinea pig. J Physiol 374, 273–288.
Cervero F & Tattersall JE (1987). Somatic and visceral inputs to the thoracic spinal cord of the cat: marginal zone (lamina I) of the dorsal horn. J Physiol 388, 383–395.
Chen J & Sandkühler J (2000). Induction of homosynaptic long-term depression at spinal synapses of sensory A-fibers requires activation of metabotropic glutamate receptors. Neuroscience 98, 141–148.[CrossRef][Medline]
Christensen BN & Perl ER (1970). Spinal neurons specifically excited by noxious or thermal stimuli: marginal zone of dorsal horn. J Neurophysiol 33, 293–307.
Craig AD, Krout K & Andrew D (2001). Quantitative response characteristics of thermoreceptive and nociceptive lamina I spinothalamic neurons in the cat. J Neurophysiol 86, 1459–1480.
Destexhe A, Neubig M, Ulrich D & Huguenard J (1998). Dendritic low-threshold calcium currents in thalamic relay cells. J Neurosci 18, 3574–3588.
Ding Y, Takada M, Shigemoto R & Mizuno N (1995). Spinoparabrachial tract neurons showing substance P receptor-like immunoreactivity in the lumbar spinal cord of the rat. Brain Res 674, 336–340.[CrossRef][Medline]
Fujino K, Koyano K & Ohmori H (1997). Lateral and medial olivocochlear neurons have distinct electrophysiological properties in the rat brain slice. J Neurophysiol 77, 2788–2804.
Galhardo V & Lima D (1999). Structural characterization of marginal (lamina I) spinal cord neurons in the cat: a Golgi study. J Comp Neurol 414, 315–333.[CrossRef][Medline]
Gauriau C & Bernard JF (2002). Pain pathways and parabrachial circuits in the rat. Exp Physiol 87, 251–258.[Abstract]
Han ZS, Zhang ET & Craig AD (1998). Nociceptive and thermoreceptive lamina I neurons are anatomically distinct. Nat Neurosci 1, 218–225.[CrossRef][Medline]
Higure Y, Katayama Y, Takeuchi K, Ohtubo Y & Yoshii K (2003). Lucifer Yellow slows voltage-gated Na+ current inactivation in a light-dependent manner in mice. J Physiol 550, 159–167.
Huguenard JR (1996). Low-threshold calcium currents in central nervous system neurons. Annu Rev Physiol 58, 329–348.[CrossRef][Medline]
Hylden JL, Anton F & Nahin RL (1989). Spinal lamina I projection neurons in the rat: collateral innervation of parabrachial area and thalamus. Neuroscience 28, 27–37.[CrossRef][Medline]
Hylden JL, Hayashi H, Bennett GJ & Dubner R (1985). Spinal lamina I neurons projecting to the parabrachial area of the cat midbrain. Brain Res 336, 195–198.[CrossRef][Medline]
Hylden JL, Hayashi H, Dubner R & Bennett GJ (1986). Physiology and morphology of the lamina I spinomesencephalic projection. J Comp Neurol 247, 505–515.[CrossRef][Medline]
Ikeda H, Heinke B, Ruscheweyh R & Sandkühler J (2003). Synaptic plasticity in spinal lamina I projection neurons that mediate hyperalgesia. Science 299, 1237–1240.
Jiang MC, Cleland CL & Gebhart GF (1995). Intrinsic properties of deep dorsal horn neurons in the L6-S1 spinal cord of the intact rat. J Neurophysiol 74, 1819–1827.
Jung HY, Staff NP & Spruston N (2001). Action potential bursting in subicular pyramidal neurons is driven by a calcium tail current. J Neurosci 21, 3312–3321.
Keay KA & Bandler R (2002). Distinct central representations of inescapable and escapable pain: observations and speculation. Exp Physiol 87, 275–279.[Abstract]
Khasabov SG, Rogers SD, Ghilardi JR, Peters CM, Mantyh PW & Simone DA (2002). Spinal neurons that possess the substance P receptor are required for the development of central sensitization. J Neurosci 22, 9086–9098.
Kozlov AS, McKenna F, Lee JH, Cribbs LL, Perez-Reyes E, Feltz A & Lambert RC (1999). Distinct kinetics of cloned T-type Ca2+ channels lead to differential Ca2+ entry and frequency-dependence during mock action potentials. Eur J Neurosci 11, 4149–4158.[CrossRef][Medline]
Light AR, Sedivec MJ, Casale EJ & Jones SL (1993). Physiological and morphological characteristics of spinal neurons projecting to the parabrachial region of the cat. Somatosens Mot Res 10, 309–325.[Medline]
Lima D & Coimbra A (1986). A Golgi study of the neuronal population of the marginal zone (lamina I) of the rat spinal cord. J Comp Neurol 244, 53–71.[CrossRef][Medline]
Lima D & Coimbra A (1989). Morphological types of spinomesencephalic neurons in the marginal zone (lamina I) of the rat spinal cord, as shown after retrograde labelling with cholera toxin subunit B. J Comp Neurol 279, 327–339.[CrossRef][Medline]
Mantyh PW, Rogers SD, Honore P, Allen BJ, Ghilardi JR, Li J, Daughters RS, Lappi DA, Wiley RG & Simone DA (1997). Inhibition of hyperalgesia by ablation of lamina I spinal neurons expressing the substance P receptor. Science 278, 275–279.
Marshall GE, Shehab SA, Spike RC & Todd AJ (1996). Neurokinin-1 receptors on lumbar spinothalamic neurons in the rat. Neuroscience 72, 255–263.[CrossRef][Medline]
McRory JE, Santi CM, Hamming KS, Mezeyova J, Sutton KG, Baillie DL, Stea A & Snutch TP (2001). Molecular and functional characterization of a family of rat brain T-type calcium channels. J Biol Chem 276, 3999–4011.
Millan MJ (2002). Descending control of pain. Prog Neurobiol 66, 355–474.[CrossRef][Medline]
Nichols ML, Allen BJ, Rogers SD, Ghilardi JR, Honore P, Luger NM, Finke MP, Li J, Lappi DA, Simone DA & Mantyh PW (1999). Transmission of chronic nociception by spinal neurons expressing the substance P receptor. Science 286, 1558–1561.
Perez-Reyes E (2003). Molecular physiology of low-voltage-activated T-type calcium channels. Physiol Rev 83, 117–161.
Prescott SA & De Koninck Y (2002). Four cell types with distinctive membrane properties and morphologies in lamina I of the spinal dorsal horn of the adult rat. J Physiol 539, 817–836.
Rogawski MA (1985). The A-current: how ubiquitous a feature of excitable cells is it?Trends Neurosci 8, 214–219.[CrossRef]
Ruscheweyh R & Sandkühler J (2002). Lamina-specific membrane and discharge properties of rat spinal dorsal horn neurones in vitro. J Physiol 541, 231–244.
Saito Y & Isa T (2000). Voltage-gated transient outward currents in neurons with different firing patterns in rat superior colliculus. J Physiol 528, 91–105.
Sandkühler J (2000). Learning and memory in pain pathways. Pain 88, 113–118.[CrossRef][Medline]
Spike RC, Puskár Z, Andrew D & Todd AJ (2003). A quantitative and morphological study of projection neurons in lamina I of the rat lumbar spinal cord. Eur J Neurosci 18, 2433–2448.[CrossRef][Medline]
Storm JF (1988). Temporal integration by a slowly inactivating K+ current in hippocampal neurons. Nature 336, 379–381.[CrossRef][Medline]
Suzuki R, Morcuende S, Webber M, Hunt SP & Dickenson AH (2002). Superficial NK1-expressing neurons control spinal excitability through activation of descending pathways. Nat Neurosci 5, 1319–1326.[CrossRef][Medline]
Swanson LW (1992). Brain Maps: Structure of the Rat Brain. Elsevier, Amsterdam.
Todd AJ, McGill MM & Shehab SA (2000). Neurokinin 1 receptor expression by neurons in laminae I, III and IV of the rat spinal dorsal horn that project to the brainstem. Eur J Neurosci 12, 689–700.[CrossRef][Medline]
Williams SR & Stuart GJ (1999). Mechanisms and consequences of action potential burst firing in rat neocortical pyramidal neurons. J Physiol 521, 467–482.
Willis WD & Coggeshall RE (1991). Sensory Mechanisms of the Spinal Cord. Plenum Press, New York.
Yarom Y, Sugimori M & Llinas R (1985). Ionic currents and firing patterns of mammalian vagal motoneurons in vitro. Neuroscience 16, 719–737.[CrossRef][Medline]
|
Acknowledgements |
|---|
Author's present address
H. Ikeda: Department of Human and Artificial Intelligence Systems, Fukui University, Fukui, Japan.
This article has been cited by other articles:
|
|
 |
|
  B. A. Graham, A. M. Brichta, and R. J. Callister Recording Temperature Affects the Excitability of Mouse Superficial Dorsal Horn Neurons, In Vitro |
