| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Journal of Physiology (2001), 536.3, pp. 785-796
© Copyright 2001 The Physiological Society
-subunit may confer benzodiazepine insensitivity to the caudal aspect of the nucleus tractus solitarii of the rat |
ABSTRACT |
|---|
 TopAbstract IntroductionMethodsResultsDiscussionReferences |
|---|
-carboline-3-carboxylate (DMCM; 1 or 10 µM), failed to depress GABA-induced currents in the cNTS.
1, 2, 2, 3 and 
2 GABAA receptor subunit mRNA in the cNTS. No alternatively spliced variants of the 1- and 2-subunits were revealed. Moreover, GABAA -subunit mRNA was found in both the cNTS and rNTS as two alternatively spliced transcripts.
-subunit-positive neurones within the cNTS with significantly fewer -subunit-positive cells in the rNTS.
-subunit in recombinant GABAA receptors may confer BZ insensitivity we propose that the paucity of BZ actions in the cNTS is due to a high level of -subunit expression. This is the first demonstration of a possible physiological impact of the -subunit on native GABAA receptors.
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
GABAA receptors possess a unique range of modulatory sites including those for benzodiazepines (BZ) and barbiturates. In many brain areas GABAA receptor-mediated responses strongly increase in the presence of drugs such as diazepam, midazolam, chlordiazepoxide or pentobarbitone (reviewed by Upton & Blackburn, 1997; Costa, 1998). Although BZ agonists and barbiturates have a number of effects in common, the pharmacology of these two groups of drugs is quite distinct. While the therapeutic ratio for barbiturates is relatively small, BZ agonists are remarkably safe drugs (Hobbs et al. 1995). For example, even high doses of midazolam, such as those used in emergency care units, seldom cause side effects related to disturbances in the autonomic nervous system (Nordt & Clark, 1997). In contrast, pentobarbitone is well known to impair autonomic control of heart rate (Stornetta et al. 1987; Barringer & Bunag, 1990).
We hypothesised that these differences might reflect a lack of BZ agonist action on GABAA-mediated inhibition in the nucleus tractus solitarii (NTS), as this would cause a major disturbance to the baroreceptor reflex. Indeed, the NTS receives and integrates information from baroreceptor afferent inputs and is crucial for numerous homeostatic reflexes. Although the NTS lacks precise viscero-topic organisation, afferents from baroreceptors, peripheral chemoreceptors, and pulmonary and gastro-intestinal receptors all terminate mainly in caudal parts of the NTS at the level of the area postrema, while gustatory afferents terminate in more rostral regions (reviewed by Blessing, 1997). For the sake of this communication we used the obex (the point at which the central canal opens into the fourth ventricle) as the boundary between the caudal NTS (cNTS) and rostral NTS (rNTS).
The presence and functional activity of GABAA receptors in the NTS is well documented: 33 % of all synaptic terminals in the NTS have been reported to contain GABA (Saha et al. 1995). The release of GABA in the NTS can be demonstrated upon K+-evoked depolarisation (Sved & Curtis, 1993). Intensive binding of [3H] SR95531, a ligand for GABAA receptors, but not for GABA uptake sites, has been described and was found to be located predominantly postsynaptically in the cNTS (Ashworth-Preece et al. 1997). Functionally, NTS GABAA receptors are involved in attenuating the baroreceptor reflex during stress (Jordan et al. 1988) and pain (Boscan & Paton, 2000). In addition, GABAA agonists such as muscimol attenuated the baroreceptor reflex when microinjected into the NTS (Okada & Bunag, 1995) and inhibited neuronal activity (Zhang & Mifflin, 1998). In contrast, blockade of GABAA receptors with bicuculline increased the activity of baroreceptive NTS neurones (Suzuki et al. 1993), indicative of some tonic inhibitory innervation. Furthermore, microinjection into the NTS of the GABA uptake blocker, nipecotic acid, caused a pressor response (Catelli et al. 1987).
Brain GABAA receptors are thought to be pentamers assembled from mainly three classes of subunit, , and (Sieghart, 1995). It is accepted that the co-expression of two types of subunit, namely an (1-3 or 5) plus 2, results in the formation of GABAA receptors sensitive to diazepam-like BZ (reviewed by Hevers & Lüddens, 1998; Mehta & Ticku, 1999). Properties of GABAA receptors have been investigated in acutely isolated NTS neurones (Nakagawa et al. 1991) but the actions of BZ have not been studied. Therefore, we sought to reveal the effects of two well-established BZ agonists, chlordiazepoxide and midazolam (an imidazobenzodiazepine), as well as those of an inverse BZ agonist, methyl 6,7-dimethoxy-4-ethyl--carboline-3-carboxylate (DMCM), on NTS GABAA receptors. For comparison, we also studied the effects of a barbiturate - pentobarbitone. In electrophysiological experiments we studied the effects of drugs on currents evoked by iontophoretically applied GABA and on inhibitory postsynaptic potentials (IPSPs) evoked by electrical stimulation within the tractus solitarii (TS). As information about the molecular make-up of GABAA receptors in the NTS is limited, we also investigated the expression of mRNA encoding six GABAA receptor subunits using the reverse transcriptase polymerase chain reaction (RT-PCR). We found in the NTS transcripts encoding the -subunit of the GABAA receptor, which might confer BZ insensitivity to GABAA receptors (Davies et al. 1997; Whiting et al. 1997). Finally, using a novel anti-GABAA- antibody (Moragues et al. 2000) we found numerous immunopositive neurones in the cNTS (but not in the rNTS) in areas which coincided exactly with the sites of the electrophysiological recordings.
Parts of the present study have been presented previously in abstract form (Kasparov & Paton, 1998, 2000; Kasparov et al. 2000).
|
METHODS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Electrophysiological experiments
Wistar rats of either sex aged between postnatal days 13 and 45 were terminally anaesthetised with halothane and decapitated. The brainstem was removed and transverse brainstem slices (350 µm thick) were cut between +700 µm and -600 µm relative to the obex in chilled, carbogen-gassed artificial cerebrospinal fluid (ACSF) using a VibroSlice (Campden Instruments, UK). Cells recorded in slices at the level of the obex and caudal to it are referred to as cNTS neurones, while those from slices rostral to the obex are referred to as rNTS neurones. The positions of the recording sites were documented on pre-drawn sections from Paxinos & Watson (1986). These sites were located medial to the TS at the same dorso-ventral extent or in a few cases in the cNTS just dorso-medial relative to the TS. The ACSF contained (mM): 10 dextrose, 125 NaCl, 24 NaHCO3, 5 KCl, 2.5 CaCl2, 1.25 MgSO4 and 1.25 KH2PO4. For recording, slices were transferred into a submerged-type chamber and superfused with ACSF (31 ± 0.5 °C) at a controlled and continuously monitored rate of 2 ml min-1. Intracellular recordings were made in the whole-cell mode using patch pipettes (3-4 M
) filled with the following solution (mM): 130 potassium gluconate, 10 Hepes, 11 EGTA, 4 NaCl, 2 MgCl2, 1 CaCl2, 2 ATP, 0.5 GTP and 5 glucose. Signals were amplified (SEC 05L, NPI, Germany) and analysed using Spike2 software (CED, UK).
Firstly, the effects of BZ, DMCM and pentobarbitone on the actions of exogenously applied GABA were studied. GABA was applied iontophoretically from a multibarrel pipette positioned blindly in close proximity to the recorded cell (similar to that used in our earlier studies; Kasparov et al. 1994; Kasparov & Paton, 1999). GABA was applied in regular cycles (1-2 s, 15-20 s interval) and a 1.5 M NaCl-filled barrel was used for current neutralisation. The cells were then voltage clamped at -80 mV (switching frequency 20 kHz, low-pass filter set to 500 Hz) so that GABAA receptor-mediated Cl- currents were inward. The GABA ejection currents (40-80 nA) were chosen to evoke submaximal responses. After the currents had remained stable for at least 5 min, drugs were bath applied for 5-6 min. Measurements were taken between the third and sixth minute. Typically, the cells were tested with a single concentration of a BZ agonist and then, after a 10-15 min washout period, pentobarbitone was applied. Two concentrations of DMCM were tested in succession in a separate group of six cNTS cells.
Secondly, the effects of BZ agonists and pentobarbitone on the actions of synaptically released GABA were studied in bridge mode. Synaptic potentials were evoked by electrical stimulation of the ipsilateral TS (0.2 ms pulse width; 0.5-5 V, 0.2-0.3 Hz). Synaptic stimuli were combined with positive current injections to allow detection of IPSPs measured at -45 to -50 mV. The evoked IPSPs in the present experiments always reversed at around -60 mV, close to the estimated reversal potential for Cl- ions based on the solutions used, and to that reported for GABAA-mediated IPSPs in the cNTS in other studies (Andresen & Mendelowitz, 1996). In our previous work, evoked IPSPs recorded from cNTS neurones were all sensitive to the GABAA receptor blocker bicuculline (Butcher et al. 1999). Therefore, in this study we did not systematically apply GABAA receptor blockers and assumed that the IPSPs were mediated by GABAA receptors. This is consistent with the paucity of glycinergic inhibition in the NTS (Jordan et al. 1988) and the ability of 100 µM pentobarbitone to potentiate all evoked IPSPs (see Results).
Chlordiazepoxide and midazolam were dissolved in ACSF. DMCM and a benzodiazepine antagonist, flumazenil, were pre-dissolved in alcohol and then added to ACSF, so that the final concentration of alcohol was below 0.1 %. For microiontophoresis a 200 mM solution of GABA (pH 5) was used. Midazolam and flumazenil (Ro 15-1788) were from Hoffmann-LaRoche. Other chemicals were obtained from Sigma-RBI (UK).
Microinjection experiments
Working heart-brainstem preparations of young rats (30-35 days old; 90-130 g) were made using recently described protocols (Paton & Kasparov, 1999). In brief, animals were deeply anaesthetised with halothane, bisected below the diaphragm, decerebrated precollicularly, and cerebellectomised to expose the fourth ventricle. The thorax and head were perfused using carbogen-gassed ACSF (as above, but containing 1.25 % Ficoll) at 31 °C. Perfusate was pumped retrogradely into the descending aorta via a double lumen catheter; the second lumen was connected to a pressure transducer. Phrenic nerve activity was recorded via a suction electrode and monitored continuously together with the electrocardiogram. Signals were acquired and analysed using Spike2 software. To study the baroreceptor reflex, the perfusion pressure was 'ramped up' at the same rate to different levels and the corresponding reflex falls in heart rate were measured before and after a bilateral (50 nl) NTS microinjection of either midazolam or pentobarbitone. The resultant falls in heart rate were analysed to calculate the baroreceptor reflex gain (i.e. 
heart rate/perfusion pressure measured at the linear part of the input-to-output reflex-response curve). Drugs were applied using pressure injection from a three-barrelled micropipette driven by a micromanipulator to 400-450 µm ventral to the dorsal surface, 200-500 µm caudal relative to the obex and between 250-500 µm from the midline. Microinjection sites were verified by post hoc histological examination of Pontamine Sky Blue deposits in fixed tissue.
RT-PCR analysis of GABAA receptor subunits in the NTS
Micropunctates of cNTS and rNTS were obtained from 450 µm thick brainstem slices prepared from 14-day-old rats as described above. Sterile glass capillaries (internal diameter ~100 µm) were used to obtain the tissue, under a high-power binocular microscope, from areas dorsomedial and medial to the TS that were comparable to those from which electrophysiological recordings were taken and which were sites of microinjection (see above). For comparison, micropunctates of neocortex were also obtained. Total RNA was then extracted using the Trizol method (Gibco), following the manufacturer's protocol.
First strand cDNAs were synthesised using 500 ng of total RNA with 1.0 µM T25V primer, 25 µM dNTP mix and displayThermo-Reverse Transcriptase (Display Systems), as per the manufacturer's instructions. PCR reactions were performed on 1/10th of the first strand cDNAs using 0.2 µM of the primers (Table 1). All PCR products were either directly sequenced or cloned before being sequenced using Big Dye terminators and AmpliTaq FS DNA polymerase (PE-Applied Biosystems) and the reactions were analysed on an ABI 377XL DNA sequencer.
Immunocytochemistry
All the details concerning the production and verification of the anti-GABA -subunit antibody, as well as the detailed immunocytochemical protocols, have been published previously (Moragues et al. 2000). In brief, rats were overdosed with halothane and perfused transcardially with 2 % paraformaldehyde and 0.2 % picric acid in 0.1 M phosphate buffer. Brains were dissected, soaked in phosphate buffer with 20 % sucrose and 30 µm sections were cut using a cryostat. Sections were incubated with the purified primary antibody (generated in rabbit; dilution 1:10 000) for 48 h at 4 °C in 0.01 M veronal buffer, containing 0.2 % Triton X-100 and 0.4 % casein. They were then incubated for 2 h at room temperature with Fc fragment-specific biotinylated goat anti-rabbit secondary antibodies (1:2000; Jackson Laboratories, Asniere, France) and for 2 h in peroxidase-conjugated streptavidin (1:2000; Jackson Laboratories).
Statistical analysis
All values quoted are the mean ± standard error of the mean (S.E.M.); n is the number of observations. Student's paired two-tailed t test and Wilcoxon's non-parametric test were applied to the raw data to reveal significant changes. Changes were interpreted as significant at the P < 0.05 probability level.
|
RESULTS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Paucity of chlordiazepoxide, midazolam and DMCM actions on cNTS neurones
Iontophoretic application of GABA readily evoked inward currents in voltage-clamped cNTS neurones (holding potential (Vh) = -80 mV; Fig. 1). In neither population of cells treated with midazolam (2 and 50 µM) or chlordiazepoxide (2 and 50 µM) did the average amplitude or integral of the GABA-induced current increase significantly (Fig. 1). Also, the holding current did not change (P > 0.1, not shown). However, in a few individual cells 50 µM chlordiazepoxide increased GABA currents: in one of seven cNTS neurones the current integral reversibly increased to 136 % of control. For comparison, 50 µM chlordiazepoxide was tested in five rNTS neurones and in two cases the GABA-evoked current integrals increased to 138 and 169 % (Fig. 1, control values = 100 %). The majority of BZ-treated cells were washed for 10 min and treated with pentobarbitone (Fig. 1). Pentobarbitone significantly potentiated GABA-evoked currents in all cNTS neurones (50 µM: 121 ± 6 %, n = 7; 100 µM: 180 ± 17 %, n = 7; P < 0.01 with Wilcoxon's test, P < 0.05 with Student's paired t test). Importantly, neither concentration of pentobarbitone changed the holding current, indicating a lack of direct opening of GABAA-operated Cl- channels (115 ± 10 % and 99 ± 3 % of control with 50 and 100 µM concentrations, respectively, P > 0.1). It should be noted that although the magnitude of the effect of pentobarbitone was different, both concentrations increased the current integral in all cNTS (n = 14) and rNTS (n = 3) neurones treated with the barbiturate without exception. In addition, 1 and 10 µM DMCM (an inverse benzodiazepine agonist) was tested in a separate group of six cNTS cells and did not attenuate the GABA current integrals (105 ± 8 % and 111 ± 8 % of control with 1 and 10 µM DMCM, respectively; Fig. 1).
 |
View larger version [in this window] [in a new window] |
|
|
Figure 1. Effect of BZ and DMCM on currents evoked by iontophoretically applied GABA in cNTS and rNTS neurones Top, chlordiazepoxide (CDZ) did not potentiate Cl- currents evoked by iontophoretically applied GABA in a cNTS neurone (left). In contrast, in 2/5 rNTS neurones tested, CDZ potentiated GABA-evoked currents (right). Pentobarbitone was effective in all NTS cells tested without exception. At -80 mV Cl- currents were inward. Note that a relatively slow rising phase of GABA-evoked current was consistently observed in these experiments; this might reflect a dendritic/diffuse location of GABAA receptors on NTS neurones. Bottom, bar charts represent average integrals of the GABA-evoked currents (means ± S.E.M.) after administration of CDZ, midazolam (MID) and DMCM. None of the drugs caused a statistically significant effect (P > 0.1 by Wilcoxon's non-parametric test and Student's paired t test applied to the raw data). By contrast, pentobarbitone increased current integrals in all NTS cells tested (P < 0.01; see Results). | ||
Of > 60 NTS neurones tested, stimulation within the TS combined with positive current injections only revealed hyperpolarising Cl--mediated IPSPs in 10 cNTS and 10 rNTS cells, consistent with our previous observations (Kasparov & Paton, 1999). IPSP integrals varied widely in different cells. As shown in Fig. 2, 50 µM chlordiazepoxide significantly increased IPSPs in a reversible (co-application of the BZ antagonist flumazenil) manner in only 2/10 cNTS cells. In contrast, the evoked IPSP was increased by 50 µM chlordiazepoxide in 5/10 rNTS cells. In spite of this trend, the overall effect of chlordiazepoxide in either part of the NTS was not significant (Wilcoxon's test, P > 0.1). In addition, midazolam (2 µM) was tested in three cNTS neurones and had no measurable effect on evoked IPSP integrals in any cell. In contrast, in all cells tested (cNTS: n = 10; rNTS: n = 8), pentobarbitone (100 µM) prolonged TS-evoked IPSPs and massively enhanced their integrals (average increase in cNTS: +736 ± 214 mV ms-1, P < 0.01; in rNTS: +440 ± 152 mV ms-1, P < 0.01). However, the difference between the effects in cNTS and rNTS was not significant (P > 0.1).
 |
View larger version [in this window] [in a new window] |
|
|
Figure 2. Changes in TS-evoked IPSP integrals in cNTS and rNTS neurones caused by 50 µM chlordiazepoxide (CDZ) Top, of 10 cNTS neurones tested, CDZ increased evoked IPSPs in only two, one of which is shown here. The CDZ effect could be antagonised by the BZ antagonist flumazenil. Some spontaneous IPSPs ( | ||
Midazolam does not affect the baroreceptor reflex circuit in cNTS
When midazolam (10 or 100 µM solutions; n = 13 and 14, respectively) was microinjected into the cNTS it caused no change in basal heart rate (P > 0.1) or baroreflex gain (Fig. 3; P > 0.1). In contrast, 100 µM pentobarbitone decreased the baroreflex gain to 53 ± 11 % of control (P < 0.01, n = 14). This effect was completely reversed after a 10 min washout. Higher concentrations of pentobarbitone (1000 µM) caused stronger and longer lasting inhibitions (data not shown). Importantly, due to the immediate spread of microinjected drugs the effect of 100 µM pentobarbitone actually relates to a lower tissue concentration. Therefore it may only result from a potentiation of endogenously released GABA (see above).
 |
View larger version [in this window] [in a new window] |
|
|
Figure 3. Pentobarbitone, but not midazolam, microinjected into the NTS suppresses the cardiac baroreceptor reflex A, a representative experiment in which the effects of midazolam and pentobarbitone, microinjected into the NTS, on the baroreceptor reflex were studied. Perfusion pressure ramps were used to evoke a baroreflex bradycardia. While 100 µM midazolam was completely ineffective, pentobarbitone (100 µM) reversibly suppressed the baroreflex. The 15 min wash period between microinjections of midazolam and pentobarbitone is not illustrated for clarity. B, pooled data indicating the lack of midazolam action (n in all cases > 12) and the highly significant and reversible action of pentobarbitone. Note that the tissue concentrations of drugs will predictably be lower than those of their solutions. | ||
Expression profiles of GABAA receptor subunits in NTS
Figure 4A illustrates the expression patterns obtained by PCR for 1-, 2-, 2-, 3- and 2S/L-subunits of the GABAA receptor, which were compared in the cNTS, rNTS and neocortex using serial dilutions of first strand cDNAs. There were no obvious qualitative differences between the cNTS, rNTS and neocortex, in that all subunits expressed in the neocortex were also present in the NTS. In particular, cNTS 1- and 2S/L-transcripts were even detected at the 10-2 dilution. To test whether some incorrectly spliced forms of the 1- or 2S/L-subunit mRNAs were present, primers were designed to span the whole length of these mRNAs (primers 1 start-stop and 2 start-stop; Table 1). The only PCR products found using cNTS cDNA had the exact sizes predicted from previously published sequences (data not shown).
 |
View larger version [in this window] [in a new window] |
|
|
Figure 4. GABAA receptor subunits in NTS A, comparison of expression of mRNA encoding | ||
Seeking a plausible explanation for the lack of BZ action in the NTS we then tested for the presence of the -subunit. Unexpectedly, two products of 459 and 381 bp were revealed using primers spanning the second transmembrane region of that subunit (Fig. 4B). Both products were cloned and sequenced and proved to belong to the published rat -subunit sequences (GenBank accession numbers: AF189262 and AF255612). However, the shorter isoform, which was prevalent in all the RT-PCR reactions from the NTS in this study, proved to be a novel splice variant of the -subunit with a 78 bp deletion in the second transmembrane region. A cDNA clone containing this variant was subsequently obtained by screening an adult rat brain Rapid Screen cDNA library (OriGene Technologies Inc.); the sequence has been deposited in GenBank (accession number AF255385). Thus, the presence of GABAA -subunit mRNA in the NTS was confirmed.
Differential expression of GABAA -subunit in cNTS and rNTS
Figure 5 illustrates representative sections stained for the GABAA -subunit. In all series (4 animals), sections taken at the level defined as the cNTS in this paper contained numerous intensely stained neurones (Fig. 5A and B). The staining appeared to be confined to the soma and proximal dendrites (note that this could, to a certain degree, depend on the particular immunocytochemical protocol used in this study). These -subunit-positive cells were much less abundant in the NTS in sections taken just rostral to the obex ('rNTS sections'), where some immunoreactivity was scattered in nuclei ventral and medial to the NTS. At the rostral-most extent of the NTS (> +700 to +800 µm) -subunit-positive cells virtually disappeared from the NTS but could still be seen in the area of the hypoglossal nerve nucleus (Fig. 5C and D).
 |
View larger version [in this window] [in a new window] |
|
|
Figure 5. Rostro-caudal gradient of GABAA A, a representative section of 'cNTS' (approximately -400 µm relative to the obex) immunostained for the GABA | ||
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The present study demonstrates that GABAA receptors located on cNTS neurones are largely insensitive to two reputed full BZ agonists, midazolam and chlordiazepoxide, and an inverse agonist, -carboline DMCM. Moreover, this area has a high level of expression of the GABAA -subunit. In contrast, in the rNTS BZ-sensitive IPSPs were fairly abundant while -subunit expression in that area was relatively low. In both the cNTS and rNTS pentobarbitone was effective in all neurones tested. This BZ insensitivity observed in electrophysiological experiments in slices was mirrored at the systems level where pentobarbitone but not midazolam suppressed the cardiac baroreceptor reflex at the level of the cNTS. To our knowledge this is the first demonstration of the impact of the -subunit on the function of native GABAA receptors in brain tissue.
In electrophysiological experiments neither chlordiazepoxide nor midazolam potentiated significantly currents evoked by GABA iontophoresis in cNTS neurones (Fig. 1). We do not believe that the concentrations of BZ used were ineffective. First, the midazolam concentration of 2 µM used in this study is similar to the effective range of concentrations used in slices by others (typically 0.1-10 µM; Jensen & Lambert, 1986; Rovira & Ben-Ari, 1993). Second, we also used 50 µM midazolam, a concentration that is much higher than that usually required to potentiate GABAA responses. Third, 50 µM chlordiazepoxide was effective in a few cells (1 of 7 in cNTS and 2 of 5 in rNTS). Finally, -carboline DMCM (at 1 or 10 µM) was also ineffective and did not diminish the GABA-evoked currents. This directly contrasts with studies in which 0.1-1 µM DMCM inhibited responses to iontophoretically applied GABA in neocortical slices (Capogna et al. 1994).
Because the pool of receptors reached by the iontophoretically administered GABA is not the same as that apposed to presynaptic release sites, we tested whether 50 µM chlordiazepoxide would potentiate IPSPs evoked by electrical stimulation of the TS (Fig. 2). The results were consistent with the data discussed above in that evoked IPSPs were not significantly enhanced by chlordiazepoxide in cNTS neurones. In two of 10 cNTS neurones tested, where to a small extent chlordiazepoxide increased the IPSP, its effect was antagonised by flumazenil. Therefore, BZ-sensitive connections are very scarce in the cNTS. Importantly, this medio-caudal aspect of the NTS, at the level of the area postrema and caudal to it, is where neurones mediating the baroreceptor reflex are located in the rat (Deuchars et al. 2000). Interestingly, some diazepam-sensitive binding of a BZ receptor ligand, [3H]Ro 15-4513, was found in the NTS with a clear rostro-caudal gradient (see Fig. 4 in Barron et al. 1997). In our study, BZ-sensitive IPSPs were found in 50 % of rNTS neurones, consistent with this gradient. In contrast to BZ, pentobarbitone potentiated exogenous or endogenous GABA responses in all NTS neurones tested in this study.
Our microinjection experiments clearly demonstrate a lack of BZ potentiation of endogenously released GABA. The working heart-brainstem preparation utilised in these experiments is decerebrate and hence devoid of anaesthesia which could complicate interpretation of data. While midazolam (up to 100 µM) was completely ineffective, 100 µM pentobarbitone faithfully and reversibly suppressed the baroreceptor reflex (higher doses caused even stronger and longer periods of inhibition). Barron et al. (1997) reported that 100 µM diazepam was able to moderately enhance the action of a GABA agonist, isoguvacine, on arterial pressure but not on the heart rate, when these drugs were microinjected into the NTS. However, the baroreceptor reflex was not tested. Importantly, the microinjections in that study were made +0.5 mm rostral to the obex, i.e. into the part of the NTS referred to as rNTS in the present work. The activity of diazepam in the rNTS would be consistent with our finding of BZ-sensitive IPSPs in that area. In contrast, strong and reliable area-independent action of pentobarbitone is consistent with previously published work indicating the depressant action of this barbiturate on autonomic homeostatic reflexes (Stornetta et al. 1987; Halliwill & Billman, 1992; Vrana et al. 1992). The effects of pentobarbitone at the concentrations used in the present work may not be due to a direct opening of the Cl- channel (see Nakagawa et al. 1991 and this study). However, the effect of pentobarbitone indicates that GABA is tonically released onto neurones mediating the baroreceptor reflex; this notion is consistent with a previous study (Suzuki et al. 1993). Thus, the lack of inhibitory action by midazolam on the baroreceptor reflex confirms that the postsynaptic GABAA receptors in the cNTS are largely BZ insensitive. This is again in line with the lack of depressant action of BZ on autonomic regulation of heart rate and blood pressure (Hobbs et al. 1995).
Brain GABAA receptors are thought to be pentamers assembled from mainly three classes of subunit, , and , with only a single -subunit per receptor (Farrar et al. 1999). The RT-PCR analysis performed in this study using micropunctates from the cNTS suggests that all the 'standard' components of BZ-sensitive GABAA receptors (1, 2, 2, 3 and 2S/L) are present. Compared with the neocortex, which is a well-established region of the brain exhibiting BZ-sensitive receptors, the abundance of all GABAA receptor subunit mRNAs in the cNTS studied in the present work appeared to be lower. However, while 1- and 2S/L-transcripts could be clearly seen at the 10-2 dilution of cNTS and rNTS templates, 2- and 2- transcripts were extremely weak even when undiluted cDNA was used (Fig. 4A). Although no quantitative comparisons can be made, the present data do demonstrate the presence of 1-, 2-, 2-, 3- and 2S/L-transcripts in both the cNTS and rNTS. Our data are consistent with information available from several previously published studies: the presence of 1-subunit mRNA in the cNTS has been demonstrated previously (Hironaka et al. 1990; Broussard et al. 1996; Durgam & Mifflin, 1998) and 3-subunit mRNA in the rNTS was revealed by in situ hybridisation (Zhang et al. 1991). Pirker et al. (2000) reported dendritic immunostaining for 1- and 2-subunits in the NTS, while faint, but distinct, staining with antibodies directed against 1-, 2-, 3-, 5-, 2/3- and 2-subunits in the NTS has been reported (Fritschy & Mohler, 1995). No clear distinction was made between frontal and caudal aspects of the nucleus (however, see Terai et al. 1998). Pirker et al. (2000; their Table 1) also indicated some 
and 3 immunostaining in the NTS but again, this appears to refer only to the most rostral aspects of the NTS at the level of the facial nucleus (see Fig. 15 in Pirker et al. 2000). In situ hybridisation data also indicated that mRNAs encoding the 6- and -subunits (which potentially could contribute to BZ insensitivity) were completely absent from the brainstem (Laurie et al. 1992). Finally, the present data are fully consistent with a recent study in which 1-, 3-, 2/3- and 2-subunits were revealed in the NTS using both RT-PCR and immunocytochemistry (Saha et al. 2001).
Thus, cNTS GABAA receptors in our experiments were largely BZ- and DMCM insensitive in spite of the presence of 1- and 2S/L-subunit mRNAs and published immunocytochemical data indicating the presence of the corresponding proteins. There was also no evidence for an alternative splicing of either of these subunits, which potentially might have affected their ability to respond to BZ. In this context, the human -subunit in combination with 1 and 2, formed GABAA receptors that were insensitive to BZ (Davies et al. 1997; Whiting et al. 1997) but sensitive to pentobarbitone (Whiting et al. 1997; Neelands et al. 1999; however, see Davies et al. 1997). We therefore tested for the presence of the -subunit in the NTS and indeed identified mRNA encoding this subunit. Surprisingly we found two mRNA species of the -subunit, one of which encodes its novel splice variant. A cDNA clone containing this latter transcript was obtained from a rat brain cDNA library (AF255385). A striking feature of it is the 26 amino acid deletion, which encompasses the second putative transmembrane domain. Such a deletion will have a major impact on the arrangement of the putative protein within the cell membrane. Clearly, its physiological significance requires further investigation. Nevertheless, the undeleted version of the -subunit mRNA is also quite abundant in the NTS. Previously it has been suggested that the -subunit in the rat is strikingly different from the other subunits due to the presence of a long repeat region (Sinkkonen et al. 2000). However, recent immunocytochemical and in situ hybridisation data (Moragues et al. 2000) suggest that the correctly spliced -subunit has no such repeat and moreover, has a much broader distribution in the brain than originally suggested (Sinkkonen et al. 2000). The splicing of the rodent -subunit seems to be unusually complicated and variants have been found both in our recent work (Moragues et al. 2000) and the present study. Expression of the -subunit in the NTS was not addressed specifically by Moragues et al. (2000), although the in situ hybridisation signal in the 'dorsal vagal complex' was very strong. Using the anti- antibody validated in that study (Moragues et al. 2000), we have revealed by immunocytochemical methods numerous -subunit-positive neurones in the cNTS (Fig. 5). These heavily stained cells could be found in all sections taken at the level of the obex and caudal to it and occupied the same location (i.e. medial to the solitary tract) as the neurones we recorded electrophysiologically. However, in more rostral areas immunopositive cells became scarce and where present were located at the boundary between the NTS and the dorsal motor nucleus of the vagus. At the rostral-most extent of the NTS (> 600-700 µm rostral to the obex) immunopositive neurones virtually disappeared (Fig. 5).
To date the features of the -subunit-containing receptors have only been studied in transfection studies where the composition of the receptor subunits is pre-set by the investigator. This study demonstrates that native GABAA receptors in the cNTS resemble those obtained in cells transfected with a combination of human 11- and -subunits (Whiting et al. 1997). It is known that 
or 
GABAA receptor subunits may 'mix' into complexes and alter the overall pharmacology of the resultant receptors (Bonnert et al. 1999; Neelands & MacDonald, 1999). Whether this applies to the native -subunit-containing receptors (and complexes are formed) or whether the -subunit actually replaces 2 in the mature receptors remains to be seen.
In conclusion, it is very likely that the BZ insensitivity in the cNTS is due to the impact of the -subunit and this insensitivity might contribute to the paucity of the deleterious effects of BZ on homeostatic cardiovascular reflexes.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| ANDRESEN, M. C. & MENDELOWITZ, D. (1996). Sensory afferent neurotransmission in caudal nucleus tractus solitarius - Common denominators. Chemical Senses 21, 387-395 | [Abstract] |
| ASHWORTH-PREECE, M., KRSTEW, E., JARROTT, B. & LAWRENCE, A. J. (1997). Functional GABAA receptors on rat vagal afferent neurones. British Journal of Pharmacology 120, 469-475 | [Abstract] |
| BARRINGER, D. L. & BUNAG, R. D. (1990). Differential anesthetic depression of chronotropic baroreflexes in rats. Journal of Cardiovascular Pharmacology 15, 10-15 | [Medline] |
| BARRON, K. W., PAVELKA, S. M. & GARRETT, K. M. (1997). Diazepam-sensitive GABAA receptors in the NTS participate in cardiovascular control. Brain Research 773, 53-60 | [Medline] |
| BLESSING, W. W. (1997). Anatomy of the lower brainstem. In The Lower Brainstem and Bodily Homeostasis, pp. 29-99. Oxford University Press, New York | |
| BONNERT, T. P., MCKERNAN, R. M., FARRAR, S., LE BOURDELLES, B., HEAVENS, R. P., SMITH, D. W., HEWSON, L., RIGBY, M. R., SIRINATHSLNGHJI, D. J. S., BROWN, N., WAFFORD, K. A. & WHITING, P. J. (1999). , a novel -aminobutyric acid type A receptor subunit. Proceedings of the National Academy of Sciences of the USA 96, 9891-9896 |
[Abstract/Full Text] |
| BOSCAN CH, P. & PATON, J. F. R. (2000). Attenuation of the baroreceptor reflex induced by nociceptive inputs is mediated by GABAA receptors in the nucleus of the solitary tract. Journal of Physiology 526.P, 131-132P | |
| BROUSSARD, D. L., LI, X. S. & ALTSCHULER, S. M. (1996). Localization of GABAA -1 mRNA subunit in the brainstem nuclei controlling esophageal peristalsis. Molecular Brain Research 40, 143-147 |
[Medline] |
| BUTCHER, J. W., KASPAROV, S. & PATON, J. F. R. (1999). Differential effects of apamin on neuronal excitability in the nucleus tractus solitarii of rats studied in vitro. Journal of the Autonomic Nervous System 77, 90-97 | |
| CAPOGNA, M., BERRETTA, N., BERTON, F., BIANCHI, R., BRUNELLI, M. & FRANCESCONI, W. (1994). The -carboline derivative DMCM decreases -aminobutyric acid responses and Ca2+-mediated K+-conductance in rat neocortical neurons in vitro. Neuropharmacology 33, 875-883 |
[Medline] |
| CATELLI, J. M., GIAKAS, W. J. & SVED, A. F. (1987). GABAergic mechanisms in nucleus tractus solitarius alter blood pressure and vasopressin release. Brain Research 403, 279-289 | [Medline] |
| COSTA, E. (1998). From GABAA receptor diversity emerges a unified vision of GABAergic inhibition. Annual Review of Pharmacology and Toxicology 38, 321-350 | [Abstract/Full Text] |
| DAVIES, P. A., HENNA, M. C., HALES, T. G. & KIRKNESS, E. F. (1997). Insensitivity to anaesthetic agents conferred by a class of GABAA receptor subunit. Nature 385, 820-823 | [Medline] |
| DEUCHARS, J., LI, Y.-W., KASPAROV, S. & PATON, J. F. R. (2000). Morphological and electrophysiological properties of neurones in the dorsal vagal complex of the rat activated by arterial baroreceptors. Journal of Comparative Neurology 417, 233-249 | [Medline] |
| DURGAM, V. R. & MIFFLIN, S. W. (1998). Comparative analysis of multiple receptor subunit mRNA in micropunches obtained from different brain regions using a competitive RT-PCR method. Journal of Neuroscience Methods 84, 33-40 | [Medline] |
| FARRAR, S. J., WHITING, P. J., BONNERT, T. P. & MCKERNAN, R. M. (1999). Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer. Journal of Biological Chemistry 274, 10100-10104 | [Abstract/Full Text] |
| FRITSCHY, J.-M. & MOHLER, H. (1995). GABAA-receptor heterogeneity in the adult rat brain: Differential regional and cellular distribution of seven major subunits. Journal of Comparative Neurology 359, 154-194 | [Medline] |
| HALLIWILL, J. R. & BILLMAN, G. E. (1992). Effect of general anesthesia on cardiac vagal tone. American Journal of Physiology 262, H1719-1724 | [Medline] |
| HEVERS, W. & LÜDDENS, H. (1998). The diversity of GABAA receptors: Pharmacological and electrophysiological properties of GABAA channel subtypes. Molecular Neurobiology 18, 35-86 | [Medline] |
| HIRONAKA, T., MORITA, Y., HAGIHIRA, S., TATENO, E., KITA, H. & TOHYAMA, M. (1990). Localization of GABAA-receptor 1 subunit mRNA-containing neurons in the lower brainstem of the rat. Molecular Brain Research 7, 335-345 |
[Medline] |
| HOBBS, R. W., RALL, T. W. & VERDON, T. A. (1995). Hypnotics and sedatives; ethanol. In Goodman and Gilman's The Pharmacological Basis of Therapeutics, ed. HARDMAN, J. G., LIMBARD, L. E., MOLINOFF, P. B., RUDDON, R. W. & GOODMAN GILMAN, A., pp. 361-396. McGraw-Hill, New York | |
| JENSEN, M. S. & LAMBERT, J. D. C. (1986). Electrophysiological studies in cultured mouse CNS neurones of the actions of an agonist and an inverse agonist at the benzodiazepine receptor. British Journal of Pharmacology 88, 717-731 | [Abstract] |
| JORDAN, D., MIFFLIN, S. W. & SPYER, K. M. (1988). Hypothalamic inhibition of neurones in the nucleus tractus solitarius of the cat is GABA mediated. Journal of Physiology 399, 389-404 | [Abstract] |
| KASPAROV, S., DAVIES, K., PATEL, U. A. & PATON, J. F. R. (2000). GABAA subunit may be responsible for the benzodiazepine (BDZ) insensitivity in the nucleus of the solitary tract (NTS) of the rat. FASEB Journal 14, A70 |
|
| KASPAROV, S. & PATON, J. F. R. (1998). GABA receptors in the nucleus of solitary tract of the developing rat are insensitive to the benzodiazepines midazolam and chlordiazepoxide. Journal of Physiology 513.P, 19-20P | |
| KASPAROV, S. & PATON, J. F. R. (1999). Differential effects of angiotensin II in the nucleus tractus solitarii of the rat - plausible neuronal mechanisms. Journal of Physiology 521, 227-238 | [Abstract/Full Text] |
| KASPAROV, S. & PATON, J. F. R. (2000). -Subunit of the GABAA receptor may be responsible for the paucity of benzodiazepine action in the nucleus of solitary tract of the rat. Journal of Physiology 523.P, 260-261P |
|
| KASPAROV, S., PAWELZIK, H. & ZIEGLGÄNSBERGER, W. (1994). Thyrotropin-releasing hormone enhances excitatory postsynaptic potentials in neocortical neurons of the rat in vitro. Brain Research 656, 229-235 | [Medline] |
| LAURIE, D. J., SEEBURG, P. H. & WISDEN, W. (1992). The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum. Journal of Neuroscience 12, 1063-1076 | [Abstract] |
| MEHTA, A. K. & TICKU, M. K. (1999). An update on GABAA receptors. Brain Research Reviews 29, 196-217 | [Medline] |
| MORAGUES, N., CIOFI, P., LAFON, P., ODESSA, M.-F., TRAMU, G. & GARRET, M. (2000). cDNA cloning and expression of a -aminobutyric acidA receptor -subunit in rat brain. European Journal of Neuroscience 12, 4318-4330 |
[Medline] |
| NAKAGAWA, T., WAKAMORI, M., SHIRASAKI, T., NAKAYE, T. & AKAIKE, N. (1991). -Aminobutyric acid-induced response in acutely isolated nucleus solitarii neurons of the rat. American Journal of Physiology 260, C745-749 |
[Medline] |
| NEELANDS, T. R., FISHER, J. L., BIANCHI, M. & MACDONALD, R. L. (1999). Spontaneous and -aminobutyric acid (GABA)-activated GABAA receptor channels formed by subunit-containing isoforms. Molecular Pharmacology 55, 168-178 |
[Abstract/Full Text] |
| NEELANDS, T. R. & MACDONALD, R. L. (1999). Incorporation of the subunit into functional -aminobutyric acidA receptors. Molecular Pharmacology 56, 598-610 |
[Abstract/Full Text] |
| NORDT, S. P. & CLARK, R. F. (1997). Midazolam: A review of therapeutic uses and toxicity. Journal of Emergency Medicine 15, 357-365 | [Medline] |
| OKADA, M. & BUNAG, R. D. (1995). Contrasting baroreflex effects of muscimol versus bicuculline injected into the nucleus tractus solitarius in anesthetized rats. Journal of Cardiovascular Pharmacology 26, 363-371 | [Medline] |
| PATON, J. F. R. & KASPAROV, S. (1999). Differential effects of angiotensin II on cardiorespiratory reflexes mediated by nucleus tractus solitarii - a microinjection study in the rat. Journal of Physiology 521, 213-225 | [Abstract/Full Text] |
| PAXINOS, G. & WATSON, C. (1986). The Rat Brain in Stereotaxic Coordinates. Academic Press Ltd, London | |
| PIRKER, S., SCHWARZER, C., WIESELTHALER, A., SIEGHART, W. & SPERK, G. (2000). GABAA receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience 101, 815-850 | [Medline] |
| ROVIRA, C. & BEN-ARI, Y. (1993). Developmental study of benzodiazepine effects on monosynaptic GABAA-mediated IPSPs of rat hippocampal neurons. Journal of Neurophysiology 70, 1076-1085 | [Medline] |
| SAHA, S., BATTEN, T. F. C. & MCWILLIAM, P. N. (1995). Glutamate, -aminobutyric acid and tachykinin-immunoreactive synapses in the cat nucleus tractus solitarius. Journal of Neurocytology 24, 55-74 |
[Medline] |
| SAHA, S., SIEGHART, W., FRITSCHY, J. M., MCWILLIAM, P. N. & BATTEN, T. F. (2001). -Aminobutyric acid receptor (GABAA) subunits in rat nucleus tractus solitarii (NTS) revealed by polymerase chain reaction (PCR) and immunohistochemistry. Molecular and Cellular Neuroscience 17, 241-257 |
[Medline] |
| SIEGHART, W. (1995). Structure and pharmacology of -aminobutyric acidA receptor subtypes. Pharmacological Reviews 47, 181-234 |
[Medline] |
| SINKKONEN, S. T., HANNA, M. C., KIRKNESS, E. F. & KORPI, E. R. (2000). GABAA receptor and subunits display unusual structural variation between species and are enriched in the rat locus ceruleus. Journal of Neuroscience 20, 3588-3595 |
[Abstract/Full Text] |
| STORNETTA, R. L., GUYENET, P. G. & MCCARTY, R. C. (1987). Autonomic nervous system control of heart rate during baroreceptor activation in conscious and anesthetized rats. Journal of the Autonomic Nervous System 20, 121-127 | [Medline] |
| SUZUKI, M., KURAMOCHI, T. & SUGA, T. (1993). GABA receptor subtypes involved in the neuronal mechanisms of baroreceptor reflex in the nucleus-tractus-solitarii of rabbits. Journal of the Autonomic Nervous System 43, 27-36 | [Medline] |
| SVED, A. F. & CURTIS, J. T. (1993). Amino-acid neurotransmitters in nucleus tractus solitarius: an in vivo microdialysis study. Journal of Neurochemistry 61, 2089-2098 | [Abstract] |
| TERAI, K., TOOYAMA, I. & KIMURA, H. (1998). Immunohistochemical localization of GABAA receptors in comparison with GABA-immunoreactive structures in the nucleus tractus solitarii of the rat. Neuroscience 82, 843-852 | [Medline] |
| UPTON, N. & BLACKBURN, T. (1997). Pharmacology of mammalian GABAA receptors. In The GABA Receptors, ed. ENNA, S. J. & BOWERY, N. J., pp. 83-120. Humana Press, Totowa, NJ, USA | |
| VRANA, M., FEJFAR, Z., HESS, L. & BLAZEK, Z. (1992). Analgosedation raises baroreflex sensitivity in acute myocardial ischaemia. Cor et Vasa 34, 421-427 | [Medline] |
| WHITING, P. J., MCALLISTER, G., VASILATIS, D., BONNERT, T. P., HEAVENS, R. P., SMITH, D. W., HEWSON, L., O'DONNELL, R., RIGBY, M. R., SIRINATHSINGHJI, D. J. S., MARSHALL, G., THOMPSON, S. A. & WAFFORD, K. A. (1997). Neuronally restricted RNA splicing regulates the expression of a novel GABAA receptor subunit conferring atypical functional properties. Journal of Neuroscience 17, 5027-5037 | [Abstract/Full Text] |
| WHITING, P. J., MCKERNAN, R. M. & IVERSEN, L. L. (1990). Another mechanism for creating diversity in -aminobutyrate type A receptors: RNA splicing directs expression of two forms of 2 subunit, one of which contains a protein kinase C phosphorylation site. Proceedings of the National Academy of Sciences of the USA 87, 9966-9970 |
[Abstract] |
| ZHANG, J.-H., SATO, M. & TOHYAMA, M. (1991). Region-specific expression of the mRNAs encoding subunits (1, 2, and 3) of GABAA receptor in the rat brain. Journal of Comparative Neurology 303, 637-657 |
[Medline] |
| ZHANG, J. & MIFFLIN, S. W. (1998). Receptor subtype specific effects of GABA agonists on neurons receiving aortic depressor nerve inputs within the nucleus of the solitary tract. Journal of the Autonomic Nervous System 73, 170-181 | [Medline] |
Acknowledgements
Financial support for this study was received from the British Heart Foundation (grants PG99055 and BS/93003) and the Royal Society.
Corresponding author
S. Kasparov: Department of Physiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.
Email: sergey.kasparov{at}bris.ac.uk
This article has been cited by other articles:
|
|
 |
|
  B. L. Jones, P. J. Whiting, and L. P. Henderson Mechanisms of anabolic androgenic steroid inhibition of mammalian {varepsilon}-subunit-containing GABAA receptors J. Physiol., June 15, 2006; 573(3): 571 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Wagner, M. P. Goldschen-Ohm, T. G. Hales, and M. V. Jones Kinetics and Spontaneous Open Probability Conferred by the {epsilon} Subunit of the GABAA Receptor J. Neurosci., November 9, 2005; 25(45): 10462 - 10468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. A. Sergeeva, N. Andreeva, M. Garret, A. Scherer, and H. L. Haas Pharmacological Properties of GABAA Receptors in Rat Hypothalamic Neurons Expressing the {epsilon}-Subunit J. Neurosci., January 5, 2005; 25(1): 88 - 95. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R.-Q. Huang and G. H. Dillon Functional Characterization of GABAA Receptors in Neonatal Hypothalamic Brain Slice J Neurophysiol, October 1, 2002; 88(4): 1655 - 1663. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) could also be seen, indicative of tonic inhibitory synaptic activity in vitro. Bottom, 10 cNTS and 10 rNTS cells, in which IPSPs were evoked in control by stimulation of TS, were first tested with 50 µM CDZ. 
indicates cells in which CDZ potentiated IPSPs in a reversible manner (by drug washout or by application of flumazenil; 2 in cNTS and 5 in rNTS). One cell in each group responded to chlordiazepoxide with a decrease in IPSPs; these are not shown for clarity. The majority of the cells were subsequently treated with pentobarbitone (50-100 µM), which consistently potentiated evoked IPSPs (see Results).
, control; 
, + midazolam or pentobarbitone; 
, wash. C, diagram drawn according to Paxinos & Watson (1986) showing the position of the dye mark (
buffer (ABgene or Toyobo), 0.5 µl 10 mM dNTP, 0.5 µl 5 M GC melt (Clontech UK), 0.5 µl sense primer (10 pmol µl-1), 0.5 µl antisense primer (10 pmol µl-1), 0.1 µl Taq DNA polymerase (5 units µl-1, ABgene), 0.5 µl KOD DNA polymerase (2.5 units µl-1, Toyobo), in a total volume of 25 µl. Cycles: 1