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. 2002 Jun 1;541(Pt 2):453–465. doi: 10.1113/jphysiol.2001.013896

Serotonin suppresses the slow afterhyperpolarization in rat intralaminar and midline thalamic neurones by activating 5-HT7 receptors

Jean-Marc Goaillard 1, Pierre Vincent 1
PMCID: PMC2290335  PMID: 12042351

Abstract

While the highest expression level of 5-HT7 receptors in the brain is observed in intralaminar and midline thalamic neurones, the physiological role of these receptors in this structure is unknown. In vivo recordings have shown that stimulation of the serotonergic raphe nuclei can alter the response of these neurones to a nociceptive stimulus, suggesting that serotonin modulates their firing properties. Using the patch-clamp technique in rat thalamic brain slices, we demonstrate that activation of 5-HT7 receptors can strongly modulate the excitability of intralaminar and midline thalamic neurones by inhibiting the calcium-activated potassium conductance that is responsible for the slow afterhyperpolarization (sAHP) following a spike discharge. This sAHP was inhibited after activation of the cAMP pathway, either by bath application of forskolin or intracellular perfusion with 8-bromo-cAMP. The inhibitory effect of 5-HT7 receptors on sAHPs was blocked by the protein kinase A antagonist RP-cAMPS. Calcium-imaging experiments showed no change in intracellular calcium levels during the 5-HT7 response, indicating that in these neurones, a global calcium signal was not necessary to activate the cAMP cascade. Finally, bath application of serotonin produced a strong increase in cytosolic cAMP concentration, as measured using the fluorescent probe FlCRhR, and an inhibition of the sAHP. Taken together, these results suggest that 5-HT7 receptors are implicated in the effect of 5-HT on sAHP in intralaminar and midline thalamic neurones, an effect that is mediated by the cAMP second-messenger cascade.

Serotonin is an important neuromodulator that controls the excitability and synaptic transmission in a wide variety of neuronal preparations. The effects of serotonin are mediated by a large family of receptors, either ionotropic or coupled to second-messenger cascades (Barnes & Sharp, 1999). Among these receptors, the most recently cloned is the 5-HT7 type, and so far its physiological function remains poorly understood.

The 5-HT7 receptor types are encoded by a single gene with three different isoforms, which may be produced by alternative splicing. In heterologous expression systems, it has been demonstrated that all three forms are positively coupled to adenylyl cyclase and have similar pharmacological profiles (Heidmann et al. 1998). The 5-HT7 receptors are activated by nanomolar concentrations of 5-carboxamidotryptamine (5-CT) or serotonin, and the agonist order of potency is 5-CT > serotonin > 8-hydroxy-2-dipropylaminotetralin. 5-HT7 receptors are blocked by low concentrations of several compounds such as risperidone, methiothepin, mesulergine, the hallucinogen lysergic acid diethylamide (commonly known as LSD), and the antipsychotic clozapine (Jasper et al. 1997; Adham et al. 1998; Thomas et al. 1998). The lack of specific pharmacology for this receptor has made difficult an unambiguous demonstration of its physiological role in the brain. Recently, specific 5-HT7 antagonists were developed, namely DR4004 (Kikuchi et al. 1999) and SB-269970 (Lovell et al. 2000), now allowing a more precise investigation of the functional role of this receptor.

In the rat brain, the relative expression of the different isoforms is constant in all regions that have been studied so far, with a large majority comprising the 5-HT7a form (Heidmann et al. 1998). During development, expression increases until postnatal day 5, then remains at the same level up to adulthood (Vizuete et al. 1997; Heidmann et al. 1998). Binding and in situ hybridization experiments have shown that the 5-HT7 receptor is present in discrete brain regions (hippocampus, hypothalamus, cortex, thalamus), with the highest expression levels occurring in the intralaminar and midline nuclei of the thalamus (Gustafson et al. 1996; Vizuete et al. 1997). In this brain region, the mRNAs coding for this receptor are six times more abundant than in the hippocampus (Heidmann et al. 1998). In spite of this high expression level, the physiological role of this receptor has never been addressed in this brain region.

The intralaminar and midline thalamic nuclei are located in the internal medullary lamina and on the midline of the thalamus (Price, 1995). Neurones in these nuclei send projections to the striatum and the cortex, and receive inputs from the superior colliculus, the spinal cord, the periacqueductal grey matter and the parabrachial nucleus (Price, 1995). They also receive projections from the raphe, indicating that serotonin may be released in these thalamic nuclei (Peschanski & Besson, 1984; Vertes et al. 1999). Intralaminar and midline thalamic neurones fire action potentials in response to nociceptive stimuli, and these nuclei are therefore considered to mediate some aspects of the pain sensation (Albe-Fessard et al. 1985; Price, 2000). Interestingly, the intensity of the response to pain in these neurones is modulated by stimulation of the raphe (Andersen & Dafny, 1983; Qiao & Dafny, 1988), suggesting that serotonin modulates the firing properties of these neurones. However, very little is known about the electrophysiological properties of intralaminar and midline thalamic neurones. While relay neurones in the principal thalamic nuclei have been studied in detail for many years, and the effects of neuromodulators on precisely identified conductances have been dissected with great care, no such extensive work has ever been performed on neurones in the intralaminar and midline nuclei.

We used the patch-clamp technique on brain slice preparations to study the effects of serotonin on the neuronal excitability of intralaminar and midline thalamic neurones. We first showed that these neurones exhibit a prominent slow afterhyperpolarization (sAHP) that lasts several seconds after a train of action potentials; this has never been observed in any principal thalamic nuclei or in the reticular thalamic nucleus. We then observed that the current underlying this sAHP is suppressed after selective activation of 5-HT7 receptors, an effect involving intracellular cAMP and protein kinase A (PKA). A preliminary report of some of these results has been presented in abstract form (Goaillard et al. 2000).

METHODS

This investigation conforms with the European Community guiding principles in the care and use of animals (86/609/CEE, CE Off J no. L358, 18 December 1986), the French decree no. 87/748 of 19 October 1987 (J Off République Française, 20 October 1987, pp. 12245-12248) and the recommendations of CNRS and University Paris VI.

Patch-clamp recordings of neurones in brain slices

Brain slices were prepared in a solution containing (mm): 125 NaCl, 2.5 KCl, 0.4 CaCl2, 1 MgCl2, 1, 25 NaH2PO4, 26 NaHCO3 and 25 glucose. Rats (9-15 days old; Janvier, France) were killed by decapitation, the brain was immersed in ice-cold solution, the thalamus was removed and coronal thalamic slices (220 μm thickness) were cut using a VT1000S vibratome (Leica, Germany). The temperature in the cutting tray of the vibratome was kept between 1 and 4 °C. Slices were kept at 32 °C for 1 h in a 100 ml beaker and then transferred to another beaker at room temperature where the calcium concentration was raised to 2 mm. All subsequent recordings were performed with this same extracellular solution. Slices were used within 6 h after cutting. Extracellular solutions were bubbled continuously with 95 % O2/5 % CO2. Patch pipette solutions contained (mm): 130 potassium methane sulphonate, 10 Hepes, 1 EGTA, 5 MgCl2, 0.1 CaCl2, 4 ATP-Na2, 5 creatine phosphate, pH 7.35, yielding ∼10 nm free calcium.

Neurones in brain slices were recorded under visual control (Edwards et al. 1989) and during the approach, a positive pressure was applied in the patch pipette to open the tissue. Release of the positive pressure and gentle suction allowed seal formation (Blanton et al. 1989). Pipettes were pulled with a vertical puller (Narishige, Japan) from borosilicate glass (Hilgenberg, Malsfeld, Germany) and had a resistance of 2-3 MΩ. Voltage- and current-clamp recordings were obtained using an Axopatch 200B (Axon Instruments, USA), digitized with an ITC16 interface (Instrutech, USA) and analysed using Axograph software (Axon Instruments). A liquid junction potential of +7.0 mV was measured and all potential values were corrected after completion of the experiment.

All experiments were carried out at room temperature (21-23 °C). All chemicals and drugs were purchased from Sigma-Aldrich-RBI except fluo-3 (Teflabs, Austin, TX, USA) and RP-cAMPS (Biolog, Bremen, Germany). Forskolin was dissolved in dry DMSO and stored at −2 0 °C in 25 mm aliquots. This stock solution was added to the extracellular solution and agitated vigorously before application. All drugs were applied in the bath, except RP-cAMPS and 8-bromo-cAMP (8-Br-cAMP). RP-cAMPS was simply added to the pipette solution. Pipette perfusion was used for intracellular delivery of 8-Br-cAMP: a solution containing 0.2-1 mm 8-Br-cAMP was ejected via a quartz capillary within the patch pipette, yielding a final concentration of 20-100 μM 8-Br-cAMP after equilibration with the pipette solution. Fluorescein (1 μM) was added to the solution ejected from the quartz capillary in order to monitor the pipette perfusion and diffusion throughout the neurone.

Statistical values are given as mean ± s.e.m., unless stated otherwise. Student's t test was used to compare two sets of data, and differences were considered significant if P < 0.05.

cAMP and calcium imaging

The fluorescent probe FlCRhR (Adams et al. 1991) was used to measure intracellular cAMP. The method used to introduce the probe into neurones in brain slices has been described elsewhere (Vincent & Brusciano, 2001). Briefly, a quartz capillary containing 0.2 μl of FlCRhR solution was placed in the tip of the patch pipette. After a gigaohm seal was obtained and the membrane patch was ruptured, the probe was ejected in the patch pipette and allowed to diffuse passively into the cytosol. The fluorescence in the cytosol was usually sufficient to start the experiment 5-15 min after release of the probe. The FlCRhR probe was prepared from recombinant PKA subunits Cα and RIIβ (gift from Professor Susan S. Taylor). The holoenzyme was purified by HPLC and concentrated to 5 μM in a solution containing 25 mm potassium phosphate, 1 mm EDTA and 5 mm β-mercaptoethanol, pH 6.8. Aliquots of 5 μl were kept at −80 °C for several years without any sign of degradation. Once thawed, aliquots were kept in the dark at 4 °C and used within a few days.

For calcium-imaging experiments, 100 μM fluo-3 was added to the pipette solution. Images were obtained with an Olympus BX50WI upright microscope with a 60 × 0.9 NA water-immersion objective. The camera was a Princeton Instruments Micromax digital camera (Roper Scientific, Trenton, NJ, USA) with a 1300 × 1030 interline CCD sensor composed of 6.7 × 6.7 μm pixels, cooled to −15 °C. A 100 W halogen lamp was used for illumination. Exposures were 1 s long for FlCRhR and 0.5 s for fluo-3. Parameters of the optical filters were: excitation D480/30; dichroic 505DCLP; emission 1 (fluorescein or fluo-3) D535/40; emission 2 (rhodamine) E560lp (all from Chroma Technology, Brattleboro, VT, USA).

RESULTS

Whole-cell current-clamp and voltage-clamp recordings were obtained from 125 thalamic neurones. The localization of the neurones in the different intralaminar and midline nuclei was determined visually with a 10 × objective. Neurones were located in the centromedian nucleus (86/125), in the paracentral nucleus (29/125) and in the intermediodorsal nucleus (10/125). No differences in the intrinsic membrane properties or pharmacological responsiveness were seen between neurones of the different nuclei, therefore all neurones recorded in this study are collectively referred to as intralaminar and midline thalamic neurones.

sAHP and intrinsic membrane properties of intralaminar and midline thalamic neurones

In current-clamp mode, intralaminar and midline neurones had a resting membrane potential of −74.9 ± 1.8 mV (n = 17) and an input resistance at −67 mV of 550 ± 56 MΩ (n = 17). Incremental depolarizing current steps in intralaminar and midline neurones triggered tonic spike firing with frequencies up to a maximum of 40 ± 4 Hz (n = 17). These discharges were followed by an afterhyperpolarization (AHP) lasting several seconds (sAHP), whose amplitude was dependent upon the number of spikes elicited by the depolarizing current (Fig. 1A). The sAHP time to peak was 1.6 ± 0.1 s (n = 17), with an average peak amplitude of −5.3 ± 0.6 mV (n = 17) and a duration always longer than 5 s.

Figure 1. Intralaminar and midline thalamic neurones express conductances responsible for a slow afterhyperpolarization (sAHP).

Figure 1

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A, in current-clamp mode, current pulses of increasing amplitude triggered trains of action potentials followed by afterhyperpolarizations (AHPs) of increasing magnitude. In this cell, the time to peak of the sAHP was 1.2 s and its duration was 9 s. The maximum amplitude was −6.3 mV, following a train of 21 spikes. A hyperpolarizing current step of −25 pA was followed by a low-threshold calcium potential crowned by sodium action potentials (inset). B, in voltage-clamp mode, a train of depolarizing pulses elicited an outward current (black, control; grey, after bath application of 100 μM cadmium). C, only the fast component of the depolarization-induced outward current was suppressed by 100 nm apamin. Traces are displayed with a faster time scale in the inset. In this cell, the time to peak of the slow component was 0.5 s and the decay time constant was 4 s, with a mean control amplitude of 26 pA. Traces shown are averages of five.

Intralaminar and midline neurones displayed a prominent low-threshold calcium spike in response to a hyperpolarizing current step (Fig. 1A, inset). This calcium spike was crowned by up to ten sodium action potentials and could be followed by an sAHP (Fig. 8A).

Figure 8. Serotonin inhibits the sAHP.

Figure 8

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A, in current-clamp mode, a current step of +175 pA induced a spike discharge (21 spikes) followed by an sAHP of −11 mV (a). A current step of −50 pA induced a rebound calcium spike crowned by seven action potentials, followed by a delayed hyperpolarization (a). The resting membrane potential of this cell was −75.5 mV and the cell was maintained at −66 mV by injecting a +20 pA tonic current. During application of 10 μM 5-HT (b), the sAHP following the spike discharge was replaced by an afterdepolarization. The sAHP following the rebound calcium spike was also suppressed, leading to a prolonged depolarization and firing for several seconds. Serotonin also changed the resting membrane potential from −75.5 to −59 mV, and the cell was then maintained at −66 mV by injecting −32 pA tonic current. B, in voltage-clamp mode, under control conditions (a), both a medium IAHP (ImAHP) and IsAHP were present. After bath application of 10 μM serotonin, IsAHP was suppressed (b, grey trace) while ImAHP remained unaffected (b, inset). Control traces are shown superimposed in black for comparison. Traces are averages of five episodes.

Recordings in voltage-clamp mode were performed in order to study the conductances underlying the sAHP. In voltage-clamp mode, the input resistance recorded at −62 mV was 730 ± 50 MΩ (n = 93). Consistent with the value of resting membrane potential obtained in current-clamp mode (-72 mV), the holding current at −62 mV generally ranged from 0 to +100 pA.

We used a protocol designed to mimic the average maximum spike discharge obtained with our current-clamp protocol (19 ± 1 spikes in 600 ms depolarization, n = 17). A train of 20 steps of 3 ms duration at a frequency of 77 Hz from a holding potential of −62 mV to +18 mV was applied regularly, with a minimal interval of 20 s. This protocol elicited a slow outward current with an average peak amplitude of 30 ± 2 pA (n = 93). The kinetic properties of this current could be determined for 85 cells, with a time to peak of 1.15 ± 0.05 s and a decay that could be fitted to a single exponential with time constant 3.63 ± 0.16 s (Fig. 1B). For the eight remaining cells, the position of the peak and the decay time constant could not be determined. This outward current is likely to be a consequence of an increase in intracellular calcium concentration following the train of depolarizations. Indeed, when calcium influx was reduced by adding 100 μM cadmium to the external medium, this current was suppressed (n = 3, Fig. 1B), while the holding current was unaffected.

In addition, 77 out of the 93 cells analysed also displayed a fast outward current of variable amplitude (mean decay time constant of 50 ± 3 ms). This fast component of the biphasic outward current was completely inhibited by 100 nm apamin (n = 4, Fig. 1C, inset; Lazdunski, 1983) and was therefore considered as a medium AHP (mAHP; reviewed in Sah, 1996; Vergara et al. 1998). In contrast, the slow component of the outward current recorded in intralaminar and midline neurones remained unaffected by apamin (Fig. 1C).

A slow outward current with similar kinetics, calcium dependence and insensitivity to apamin has been described in several other preparations (Sah, 1996; Vergara et al. 1998); thus, the slow outward current present in intralaminar neurones will be hereafter referred to as IsAHP. Whether recorded in current-clamp or voltage-clamp mode, IsAHP was a distinctive feature of the intralaminar and midline neurones, since it was recorded in 88 % of them (110/125). This is in sharp contrast with what has been reported for all other thalamic neurones (Steriade et al. 1997) and prompted us to characterize further the modulation of this sAHP. Cells that presented no IsAHP were not analysed further.

Activation of PKA suppresses IsAHP

A characteristic feature of IsAHP is its sensitivity to PKA. It has been shown in several preparations that activation of PKA, either pharmacologically or following neuromodulator application, leads to a strong decrease in IsAHP amplitude (Nicoll, 1988; Dunwiddie et al. 1992; Haas & Gähwiler, 1992; Pedarzani & Storm, 1993; Haug & Storm, 2000). In our preparation, for all cells presenting IsAHP, bath application of 12.5 μM forskolin decreased the IsAHP amplitude by 86 ± 3 % (n = 6; Fig. 2A, B and Fig. 3C, D). This inhibition was associated with a decrease in the outward holding current recorded at −62 mV (from +66 ± 11 pA to +44 ± 7 pA, n = 6) and a decrease in the input conductance of the cell (-760 ± 210 pS, n = 6).

Figure 2. Forskolin and 8-bromo-cAMP (8-Br-cAMP) inhibit the sAHP.

Figure 2

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A, the current associated with the sAHP, termed IsAHP, before (a) and after (b) bath application of 12.5 μM forskolin in a paracentral neurone. Forskolin decreased IsAHP and reduced the tonic outward current. B, time course of changes in IsAHP amplitude: forskolin application reduced IsAHP to 5 % of the control amplitude. C, sAHP measured in current-clamp mode was inhibited by intracellular perfusion with 8-Br-cAMP: a, control; the resting membrane potential was −67.5 mV; b, after ejection into the patch pipette of a solution containing 200 μM 8-Br-cAMP yielding a final concentration of 20 μM after equilibration with the pipette solution, the sAHP was inhibited and the resting membrane potential was −57 mV. The cell was maintained at −67.5 mV by injecting −30 pA of current. The control trace is shown superimposed for comparison. D, time course of changes in the sAHP during 8-Br-cAMP perfusion. Traces in A and C are averages of the five episodes indicated by the square brackets in B and D, respectively.

Figure 3. IsAHP is inhibited following activation of 5-HT7 receptors.

Figure 3

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Pindolol (10 μM) was present during all of these experiments. A, IsAHP recorded in a centromedian neurone. After bath application of 10 nm 5-carboxamidotryptamine (5-CT; b), the IsAHP amplitude fell to 13 % of the control value (a) and recovered to 35 % of the control amplitude 20 min after removing the drug (c). B, time course of changes in IsAHP amplitude during 5-CT application. C, in the continuous presence of 1 μM SB-269970 (a 5-HT7 antagonist), 10 nm 5-CT had no effect (b). In this particular cell, application of 200 nm 5-CT was tried and may have reduced IsAHP (c), competing with SB-269970. Forskolin (FSK) at a concentration of 12.5 μM reduced IsAHP to 20 % of the control value. D, time course of changes in IsAHP amplitude. Traces in A and C are averages of the five episodes indicated by the square brackets in B and D, respectively.

Since forskolin has some unspecific pharmacological effects (Boutjdir et al. 1990; Leidenheimer et al. 1991), PKA was activated directly using the non-hydrolysable cAMP analogue 8-Br-cAMP. This compound was delivered into the cytosol of the recorded cell using the patch-pipette perfusion technique (see Methods): this ensures that 8-Br-cAMP exerts its effects only in the recorded cell and does not affect surrounding neurones. Moreover, this technique allowed recording of the sAHP under control conditions before ejection of the 8-Br-cAMP from the tip of the patch pipette. In the three cells tested with this protocol, the sAHP was totally suppressed 10 min after ejection of the solution containing 8-Br-cAMP (Fig. 2C, D). This inhibition was associated with a depolarization of the resting membrane potential (from −72 mV to −57 mV, n = 3). However, the first component of the AHP after the spike discharge remained unchanged, consistent with the lack of modulation of the mAHP by intracellular cAMP (Fig. 2C). These experiments show that the sAHP in intralaminar and midline thalamic neurones is sensitive to PKA activation, as described previously in the hippocampus (Nicoll, 1988; Dunwiddie et al. 1992; Haas & Gähwiler, 1992; Pedarzani & Storm, 1993; Haug & Storm, 2000).

Selective activation of 5-HT7 receptors strongly inhibits IsAHP

5-HT7 receptors are highly expressed in the intralaminar and midline nuclei. Since when expressed in heterologous systems these receptors appear to be positively coupled to adenylyl cyclase, we supposed that activation of these receptors in intralaminar and midline neurones could lead to a decrease in IsAHP. So far, there is no specific agonist for 5-HT7 receptors. However, at nanomolar concentrations, 5-CT only activates 5-HT1, 5-HT5B and 5-5-HT7 receptors (Barnes & Sharp, 1999). 5-HT5B receptors are not expressed in intralaminar and midline nuclei (Branchek & Zgombick, 1997) and 10 μM pindolol was used throughout the following experiments in order to block the 5-HT1 receptors (Barnes & Sharp, 1999).

Application of 10 nm 5-CT induced a mean inhibition of 78 ± 5 % of IsAHP (n = 9, Fig. 3A, B). This effect partially recovered after times longer than 25 min. Increasing the 5-CT concentration to 50 nm and 100 nm did not significantly increase the inhibition of IsAHP (82 ± 2 %, n = 7, and 85 ± 6 %, n = 8, respectively; Fig. 5). This effect was not significantly different from the inhibition obtained after direct activation of adenylyl cyclase with forskolin (Fig. 5), and was paralleled by a decrease in the tonic outward current (-13 ± 2 pA for all 5-CT concentrations, n = 24) and in the input conductance of the cell (-390 ± 70 pS, n = 24).

Figure 5. Modulation of IsAHP in different situations.

Figure 5

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The error bars indicate s.e.m. The number of cells from which data were obtained under each condition is given above each bar.

The novel 5-HT7 receptor antagonist SB-269970 (Lovell et al. 2000) was used to confirm further the involvement of the 5-HT7 receptor in the modulation of IsAHP by 5-CT. Bath application of 1 μM SB-269970 had no effect on either the IsAHP or the holding current. However, applications of 10 nm 5-CT in the presence of 1 μM SB-269970 inhibited the IsAHP by only 15 ± 5 % (n = 6; Fig. 3C, D and Fig. 5), indicating that most of the 5-CT effect on IsAHP involves the 5-HT7 receptor. In addition, no change in the tonic outward current was observed under these pharmacological conditions (+1.3 ± 1.6 pA, n = 6).The inhibitory effect of forskolin on IsAHP persisted in the presence of 1 μM SB-269970 (Fig. 3C, D), showing that SB-269970 does not prevent IsAHP inhibition by the cAMP cascade. At a lower dose (200 nm), SB-269970 was still able to block the effect of 10 nm 5-CT, which decreased IsAHP by only 26 ± 9 % (n = 3, Fig. 5).

The effect of 5-HT7 receptor activation on IsAHP involves PKA

In order to demonstrate that activation of the cAMP cascade is necessary for the modulation of IsAHP by 5-HT7 receptors, 100 μM RP-cAMPS was added to the intracellular recording solution. Before applying 5-CT, the whole-cell configuration was maintained for at least 25 min to allow for the diffusion of the compound into the fine processes. Under these conditions, the inhibition of IsAHP by 10 nm 5-CT was reduced to 30 ± 2 % (n = 6; Fig. 4), which is significantly different from what was obtained in the absence of RP-cAMPS (Fig. 5). No change in the tonic outward current was measured under these conditions (-1.5 ± 2.5 pA, n = 6).

Figure 4. Intracellular dialysis with the protein kinase A inhibitor RP-cAMPS reduced the effect of 5-CT on IsAHP.

Figure 4

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The neurone was recorded for 20 min in the whole-cell configuration with a pipette solution containing 100 μM RP-cAMPS. Aa, control; Ab, after bath application of 10 nm 5-CT. B, time course of changes in IsAHP amplitude. Traces in Aa and b are averages of the five episodes indicated by the square brackets (labelled a and b, respectively) in B.

5-HT7-mediated IsAHP inhibition does not involve changes in intracellular calcium concentration

In heterologous expression systems and cultured hypothalamic neurones, activation of 5-HT7 receptors has been shown to trigger an increase in intracellular calcium concentration lasting several minutes, which activates calcium-dependent adenylyl cyclases 1 or 8 (Baker et al. 1998). The genes coding for these adenylyl cyclases are expressed in the thalamus (Cali et al. 1994; Matsuoka et al. 1994, 1997), and the question arises whether an increase in intracellular calcium concentration could be the initial step in the 5-HT7 response in intralaminar and midline neurones. To test this possibility, we could not buffer the intracellular calcium since this would have led to a complete block of the sAHP. Instead, we decided to measure directly changes in intracellular calcium concentration with fluo-3 while simultaneously recording IsAHP (Fig. 6). Under control conditions, the IsAHP protocol induced an increase in fluorescence intensity, indicating an increase in intracellular calcium concentration (Fig. 6C, D). The amplitude and kinetics of this calcium signal were dependent upon the cellular location, the fluorescence changes being faster in dendrites.

Figure 6. Intracellular calcium is not affected by 5-CT.

Figure 6

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A, the neurone was loaded with 100 μM fluo-3 via the patch pipette. The shape on the left of the image is the patch pipette. The fluorescence was monitored on the cell body and on a dendrite, as shown by the outlines drawn on the micrograph. B, IsAHP was recorded using the standard protocol under control conditions and after bath application of 100 nm 5-CT. Fluorescence was quantified simultaneously on images acquired during this protocol, on the dendrite region (C) and on the cell body (D), with F the fluorescence at the beginning of the episode and ΔF the fluorescence change during the episode. Each data point corresponds to one image. E, time course of changes in the IsAHP amplitude during 5-CT application. F and G, during the course of this experiment two images were recorded for each IsAHP protocol, one before triggering the train of depolarization (baseline) and one at the peak of the fluorescence response, as determined in C and D (peak). F indicates the fluorescence measured on the dendritic region and G indicates the fluorescence on the cell body given in counts per pixel per 0.5 s exposure. The comparative time course of changes in IsAHP and fluorescence shown in B, C and D was determined at the times indicated by Inline graphicand ⋆ in G.

The IsAHP protocol was applied to neurones with maximal intervals of 34 s, while recording baseline fluorescence and fluorescence at the peak of the calcium signal. Bath application of 100 nm 5-CT strongly reduced IsAHP (Fig. 6B, E), while no significant change in either the baseline or peak fluorescence signals was observed (Fig. 6C, D, F, G). The same result was obtained from all five cells tested with this protocol. These experiments show that 5-HT7 receptor activation does not induce any detectable change in baseline calcium levels that could be correlated to suppression of the IsAHP. In these neurones, a global increase in intracellular calcium concentration was not necessary for obtaining a PKA-mediated effect following the activation of 5-HT7 receptors.

This experiment also shows that 5-CT induces no major change in the peak calcium signal elicited by the IsAHP protocol. The small change in ΔF/F values is attributable to a slow and continuous drift of baseline fluorescence during the course of the experiment, and 5-CT application did not produce a strong enough decrease in the calcium signal to account for the suppression of IsAHP.

Serotonin reproduces the 5-HT7-mediated effects on intralaminar and midline neurones

Intralaminar neurones express other types of serotonin receptors, and at least 5-HT1A receptors are present in these neurones. Since 5-HT1A receptors are negatively coupled to adenylyl cyclase, the release of serotonin in intralaminar nuclei might have ambiguous effects. In order to determine the net effect of serotonin in these neurones, we studied the effects of bath application of serotonin in the absence of any receptor antagonist.

The fluorescent probe FlCRhR (Adams et al. 1991) allows a direct measurement of increases in intracellular cAMP concentration in real time during a drug application. The probe was introduced into the cytosol via the patch pipette. Bath application of 10 μM serotonin increased the ratio in eight cells (ratio change = 6.6 ± 2 %; Fig. 7), while five other cells failed to respond. A response was considered as positive when serotonin produced a ratio change greater than 2 %, well above the baseline fluctuation. Cells that did not show a ratio increase upon serotonin application should not be taken as an indication of a lack of cAMP response, since it has already been shown in previous experiments that not all neurones display a ratio increase in response to stimulation of adenylyl cyclase (Vincent & Brusciano, 2001). In order to compare the time course of changes in cAMP and IsAHP, it would have been very interesting to record simultaneously both parameters. However, in the presence of FlCRhR in the cytosol, trains of depolarizations did not induce any IsAHP. This is probably due to the presence of some free catalytic subunits in the probe. Constitutive PKA activity of the probe has already been reported with the L-type calcium current in cardiomyocytes (Goaillard et al. 2001). The absolute calibration of the ratio change in terms of intracellular cAMP concentration could not be performed because resting ratio values were quite variable from cell to cell and depended strongly upon loading of the dye by diffusion through the pipette tip, as discussed previously (Vincent & Brusciano, 2001).

Figure 7. 5-HT increases intracellular cAMP.

Figure 7

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After the fluorescent probe FlCRhR diffused from the pipette (left of the cell) into the recorded neurone (A), pairs of images were acquired at the emission wavelengths of fluorescein and rhodamine. In the pseudocolour images (B), the ratio of fluorescein over rhodamine emission is indicated by the hue, and the intensity of the original image is indicated by the intensity, as shown on the calibration square. Intensity values are in counts s−1 pixel−1. Ratio values are arbitrary units. C, time course of changes in the ratio quantified on the cell body during the application of 10 μM 5-HT. A gamma correction of 0.5 was applied to A in order to enhance the visibility of fine neurites.

However, these experiments demonstrate undoubtedly that serotonin is able to activate the cAMP cascade in most, if not all cells. This shows a strong prevalence of Gs-coupled receptors versus Gi-coupled receptors in the response to serotonin. This was further confirmed by electrophysiological recordings where, in all neurones tested, bath application of 10 μM serotonin induced a strong inhibition of sAHP or IsAHP (94 ± 4 %, n = 5, Fig. 8). In current-clamp mode, this inhibition resulted in a conversion of the AHP to an afterdepolarization following the spike discharge and could also result in a loss of repolarization after the calcium rebound (Fig. 8A). Voltage-clamp recordings showed that the fast component of the IsAHP was unaffected by the application of 10 μM 5-HT, consistent with the results obtained with 5-CT application (Fig. 8Bb, inset). Inhibition of the sAHP was associated with a depolarization of the resting membrane potential and inhibition of IsAHP, with a concomitant decrease in the tonic outward current and in the input conductance of the cell (-270 ± 100 pS, n = 5).

DISCUSSION

Electrophysiological properties of intralaminar and midline thalamic neurones

In the report presented here, we describe an sAHP in the centromedian, paracentral and intermediodorsal thalamic nuclei. This sAHP was blocked when the influx of calcium was reduced, was insensitive to apamin and was suppressed following activation of PKA. sAHPs sharing these properties have been described in various neuronal types but until now have never been observed in thalamic reticular or sensory nuclei (Steriade et al. 1997). Therefore, since previous intracellular recordings in the intralaminar parataenial nucleus of the guinea-pig also revealed an sAHP (McCormick & Prince, 1988), it seems that the presence of an sAHP is a distinctive feature of intralaminar and midline neurones. In addition to the sAHP, most intralaminar and midline thalamic neurones displayed an apamin-sensitive mAHP, with same characteristics as described in the thalamic reticular nucleus (Avanzini et al. 1989; Bal & McCormick, 1993; Debarbieux et al. 1998) and in several other preparations (Sah, 1996).

Interestingly, the input resistance of intralaminar and midline thalamic neurones was almost three times higher than that recorded under identical conditions in the relay neurones of thalamic sensory nuclei (258 ± 20 MΩ, n = 13). A high input resistance in another intralaminar nucleus (parataenial nucleus) was also reported using intracellular recordings (McCormick & Prince, 1988). An IsAHP of relatively small amplitude as recorded under our conditions is thus sufficient to affect strongly the membrane potential, and indeed, current-clamp recordings show a prominent sAHP. This sAHP could be elicited by either a train of action potentials or by a low-threshold calcium spike. The presence of this sAHP could have several consequences on the electrical behaviour of intralaminar and midline thalamic neurones. First, if the neurone is firing tonically, the sAHP would hyperpolarize the cell and reduce the firing rate. Second, if the cell is in bursting mode, the sAHP would help to deinactivate the low-threshold calcium current and thus participate in the production of rhythmic activity.

In addition, a decrease of IsAHP was always associated with a depolarization (in current clamp) or an inward shift of the holding current (in voltage clamp). This was observed when the cAMP cascade was activated directly by forskolin or 8-Br-cAMP, or when 5-HT7 receptors were activated by either 5-CT or by serotonin. Since the depolarizing current Ih is modulated by cAMP, it was suspected initially that this conductance mediates the depolarization or inward current. However, the inward shift of the holding current was always associated with a decrease in membrane conductance, showing that this effect is the result of a reduction in an outward conductance, and that Ih is not involved. Another possibility is that the tonic outward current is mediated by the continuous activation of the channels responsible for IsAHP. This tonic outward current was not affected by bath-applied cadmium, ruling out the involvement of a calcium influx via a window calcium current in the tonic activation of IsAHP. However, one might still speculate that the IsAHP conductances are tonically active even in the absence of intracellular calcium. Alternatively, a totally unrelated potassium conductance may underlie this tonic outward current, such as one of the many 2-P-domain channels, many of which are modulated by PKA (Goldstein et al. 2001). This current may also be related to the leak potassium current already described in thalamic sensory relay neurones (Steriade et al. 1997). The precise characterization of this outward current clearly awaits further investigation.

5-HT7 receptors inhibit sAHP via the cAMP cascade

Serotonin is one of the neuromodulators that participate in the control of thalamic excitability. Since intralaminar and midline neurones express the highest amounts of 5-HT7 mRNA in the brain, it was important to determine whether activation of this receptor could have physiological effects on neurones in this structure. Our results show that intralaminar and midline thalamic neurones express fully functional 5-HT7 receptors, since selective activation of this receptor has a powerful inhibitory effect on IsAHP, and this inhibition is completely blocked by the recently developed specific antagonist SB-269970 (Lovell et al. 2000). This effect is consistent with results obtained recently with CA3 pyramidal neurones in the hippocampus, where activation of 5-HT7 receptors also inhibited the sAHP (Bacon & Beck, 2000). However, in our experiments the effects of 5-CT did not recover. This may be a consequence of our recording conditions, since whole-cell recording leads to dialysis of intracellular compounds that may be needed to reverse the 5-HT7-mediated effect.

5-HT7 receptors expressed in heterologous systems are positively coupled to adenylyl cyclase (Vanhoenacker et al. 2000) and thus it was suspected that the cAMP cascade mediates the inhibition of the sAHP in intralaminar neurones. In the anterodorsal thalamus, it was demonstrated that 5-HT7 receptors were positively coupled to adenylyl cyclase, since activation of these receptors shifts the Ih activation curve, an effect occluded by intracellular perfusion with cAMP (Chapin & Andrade, 2001). In our experiments, the effect of 5-CT on the sAHP in intralaminar and midline thalamic neurones was mimicked by direct activation of adenylyl cyclase with forskolin, or following intracellular application of 8-Br-cAMP. Furthermore, the inhibitory effect of 5-CT was blocked when the cell was filled with the highly specific PKA inhibitor RP-cAMPS. These experiments demonstrate clearly that the inhibitory effect of 5-HT7 receptor on the sAHP is mediated by PKA.

One report has indicated that the activation of 5-HT7 receptors first triggers a global increase in cytosolic calcium concentration which, in a second step, activates calcium-dependent adenylyl cyclase types 1 and 8 (Baker et al. 1998). This was observed in heterologous expression systems as well as in primary cultures of hypothalamic and hippocampal neurones. Calcium imaging in our preparation revealed that 5-CT produced no change in baseline calcium levels, neither in the soma nor in the dendrites, where a high surface vs. volume ratio should favour the detection of submembrane calcium signals. At the same time, IsAHP was recorded and displayed the expected inhibition upon 5-CT application. As a control, the global calcium signal triggered by the IsAHP protocol was measurable with fluo-3, and was observed to affect the membrane since it activated IsAHP. However, these calcium signals were unable to raise intracellular cAMP, since IsAHP displayed no activity-dependent decrease. In conclusion, 5-HT7 receptors do not seem to couple to adenylyl cyclases via a calcium signal affecting the cytosol, and a direct coupling via Gs proteins seems more likely, as has been reported to occur in other heterologous expression systems (Vanhoenacker et al. 2000).

The calcium-imaging experiment also indicated that 5-CT does not affect the peak of the calcium signal elicited by the IsAHP protocol. Although this data would need more precise quantification using ratiometric indicators, it is consistent with previous work showing that the inhibition of IsAHP by neuromodulators is not a consequence of an inhibition of calcium influx into the cytosol, but is rather due to an inhibition of the calcium-activated potassium conductances (Nicoll, 1988; Sah & Clements, 1999; Lancaster & Batchelor, 2000).

Physiological effects of serotonin

Since intralaminar and midline thalamic neurones express other types of 5-HT receptors, such as 5-HT1A, 5-HT2A, 5-HT5A and 5-HT6 (Branchek & Zgombick, 1997; Barnes & Sharp, 1999), what is the net physiological effect of serotonin released in this part of the thalamus? For example, 5-HT1 receptors activate Gi/o proteins, which may counteract the 5-HT7-mediated stimulation of adenylyl cyclase. Our imaging experiments with the cAMP probe FlCRhR revealed that serotonin can produce strong increases in intracellular cAMP concentration. In parallel, voltage- and current-clamp recordings showed that serotonin also suppressed the sAHP and a tonic outward current. Thus, serotonin reproduces the effects of direct activation of the cAMP cascade or specific activation of 5-HT7 receptors, showing that stimulation of all 5-HT receptors present on these neurones has the same effect as specific activation of 5-HT7 receptors. This indicates that 5-HT7 receptors play an essential role in mediating serotonin responses in these neurones and that pharmacological activation or inactivation of this receptor may have considerable physiological consequences.

The inhibitory effect of serotonin on the sAHP has important consequences on the firing properties of these neurones: after serotonin application, repolarization of the low-threshold calcium action potential is delayed and tonic firing is facilitated. Tonic firing is also facilitated by depolarization of the resting membrane potential following the inhibition of the tonic outward current. In the parataenial nucleus, a similar effect of noradrenaline has been reported: noradrenaline suppresses the sAHP and depolarizes the cell, resulting in an increased firing (McCormick & Prince, 1988). It would be interesting to check if this response also involves the cAMP cascade.

Our results show the presence of a so far unsuspected mechanism for modulating neuronal excitability in the thalamus, since in intralaminar and midline thalamic neurones, the target of the modulation is the sAHP instead of the well-characterized Ih. In sensory relay neurones, neuromodulators such as noradrenaline, serotonin and histamine increase Ih via activation of the cAMP cascade (McCormick & Pape, 1990; McCormick & Williamson, 1991), while adenosine or GABA decrease Ih by inhibiting cAMP production (Pape, 1992). Our results show that in intralaminar and midline thalamic nuclei, serotonin also produces an increase in cAMP, but the physiological effector is quite different, being the inhibition of the sAHP and a still uncharacterized tonic outward current. However, in both thalamic regions, the effect of an increase in cAMP converges to the same result, which is a depolarization and an increase in the excitability of the cell.

A physiological effect of serotonin on the neuronal excitability of intralaminar and midline thalamic neurones was suggested by extracellular recordings performed in vivo (Qiao & Dafny, 1988): for a population of neurones responding to painful stimulation by an increased firing frequency, raphe stimulation increased the spontaneous discharge as well as the amplitude of the response to painful stimuli. This is consistent with the 5-HT7-mediated increase in excitability we observed in our preparation.

In conclusion, in the study presented here we have shown that the 5-HT7 receptors expressed in the intralaminar and midline thalamic neurones are able to induce a powerful modulation of neuronal excitability via the inhibition of the conductance responsible for the sAHP. As a consequence, 5-HT7 receptors in intralaminar and midline thalamic neurones may be an important target in therapeutics. Several drugs used in neurology and psychiatry have a high affinity for 5-HT7 receptors. Antipsychotic drugs such as clozapine have been reported to bind to 5-HT7 receptors with nanomolar affinity (Shen et al. 1993; Adham et al. 1998). Whether the intralaminar and midline thalamic nuclei are involved in psychiatric disorders should thus be re-evaluated. A more striking observation is that several tricyclic antidepressants such as amitriptyline also bind to the 5-HT7 receptor with a high affinity (Shen et al. 1993). In addition to their antidepressant effect, these molecules are used at lower doses to treat chronic pain (Bryson & Wilde, 1996), particularly that generated non-peripherally (Pilowsky & Barrow, 1990), which is thought to involve the intralaminar and midline thalamic nuclei (Albe-Fessard et al. 1985; Dostrovsky & Guilbaud, 1990; Price, 2000). The central place that 5-HT7 receptors have in the control of excitability in intralaminar thalamic nuclei raises the hope of finding new specific anti-nociceptive drugs that may act selectively on this receptor.

Acknowledgments

We thank Roger Y. Tsien and Stephen R. Adams for their support in the preparation of FlCRhR. We would also like to thank Nathalie Leresche for helpful comments on the manuscript. This work was supported by a grant from the Fondation pour la Recherche Médicale.

REFERENCES

  1. Adams SR, Harootunian AT, Buechler YJ, Taylor SS, Tsien RY. Fluorescence ratio imaging of cyclic AMP in single cells. Nature. 1991;349:694–697. doi: 10.1038/349694a0. [DOI] [PubMed] [Google Scholar]
  2. Adham N, Zgombick JM, Bard J, Branchek TA. Functional characterization of the recombinant human 5-hydroxytryptamine7(a) receptor isoform coupled to adenylate cyclase stimulation. Journal of Pharmacology and Experimental Therapeutics. 1998;287:508–514. [PubMed] [Google Scholar]
  3. Albe-Fessard D, Berkley KJ, Kruger L, Ralston HJ, 3, Willis WD., Jr Diencephalic mechanisms of pain sensation. Brain Research. 1985;356:217–296. doi: 10.1016/0165-0173(85)90013-x. [DOI] [PubMed] [Google Scholar]
  4. Andersen E, Dafny N. Dorsal raphe stimulation reduces responses of parafascicular neurons to noxious stimulation. Pain. 1983;15:323–331. doi: 10.1016/0304-3959(83)90069-6. [DOI] [PubMed] [Google Scholar]
  5. Avanzini G, De Curtis M, Panzica F, Spreafico R. Intrinsic properties of nucleus reticularis thalami neurones of the rat studied in vitro. Journal of Physiology. 1989;416:111–122. doi: 10.1113/jphysiol.1989.sp017752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bacon WL, Beck SG. 5-Hydroxytryptamine(7) receptor activation decreases slow afterhyperpolarization amplitude in CA3 hippocampal pyramidal cells. Journal of Pharmacology and Experimental Therapeutics. 2000;294:672–679. [PubMed] [Google Scholar]
  7. Baker LP, Nielsen MD, Impey S, Metcalf MA, Poser SW, Chan G, Obrietan K, Hamblin MW, Storm DR. Stimulation of type 1 and type 8 Ca2+/calmodulin-sensitive adenylyl cyclases by the Gs-coupled 5-hydroxytryptamine subtype 5-HT7A receptor. Journal of Biological Chemistry. 1998;273:17469–17476. doi: 10.1074/jbc.273.28.17469. [DOI] [PubMed] [Google Scholar]
  8. Bal T, McCormick DA. Mechanisms of oscillatory activity in guinea-pig nucleus reticularis thalami in vitro: a mammalian pacemaker. Journal of Physiology. 1993;468:669–691. doi: 10.1113/jphysiol.1993.sp019794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Barnes NM, Sharp T. A review of central 5-HT receptors and their function. Neuropharmacology. 1999;38:1083–1152. doi: 10.1016/s0028-3908(99)00010-6. [DOI] [PubMed] [Google Scholar]
  10. Blanton M, Lo Turco JJ, Kriegstein A. Whole-cell recording from neurons in slices of reptilian and mammalian cerebral cortex. Journal of Neuroscience Methods. 1989;30:203–210. doi: 10.1016/0165-0270(89)90131-3. [DOI] [PubMed] [Google Scholar]
  11. Boutjdir M, Méry PF, Hanf R, Shrier A, Fischmeister R. High affinity forskolin inhibition of L-type Ca2+ current in cardiac cells. Molecular Pharmacology. 1990;38:758–765. [PubMed] [Google Scholar]
  12. Branchek TA, Zgombick JM. Molecular biology and potential functional role of 5-HT 5, 5-HT 6 and 5-HT 7 receptors. In: Baumgarten HG, Gothert M, editors. Serotoninergic Neurons and 5-HT Receptors in the CNS. Berlin: Springer-Verlag; 1997. pp. 475–497. [Google Scholar]
  13. Bryson HM, Wilde MI. Amitriptyline. A review of its pharmacological properties and therapeutic use in chronic pain states. Drugs and Aging. 1996;8:459–476. doi: 10.2165/00002512-199608060-00008. [DOI] [PubMed] [Google Scholar]
  14. Cali JJ, Zwaagstra JC, Mons N, Cooper DM, Krupinski J. Type VIII adenylyl cyclase. A Ca2+/calmodulin-stimulated enzyme expressed in discrete regions of rat brain. Journal of Biological Chemistry. 1994;269:12190–12195. [PubMed] [Google Scholar]
  15. Chapin EM, Andrade R. A 5-HT(7) receptor-mediated depolarization in the anterodorsal thalamus. II. Involvement of the hyperpolarization-activated current I(h) Journal of Pharmacology and Experimental Therapeutics. 2001;297:403–409. [PubMed] [Google Scholar]
  16. Debarbieux F, Brunton J, Charpak S. Effect of bicuculline on thalamic activity: a direct blockade of IAHP in reticularis neurons. Journal of Neurophysiology. 1998;79:2911–2918. doi: 10.1152/jn.1998.79.6.2911. [DOI] [PubMed] [Google Scholar]
  17. Dostrovsky JO, Guilbaud G. Nociceptive responses in medial thalamus of the normal and arthritic rat. Pain. 1990;40:93–104. doi: 10.1016/0304-3959(90)91056-O. [DOI] [PubMed] [Google Scholar]
  18. Dunwiddie TV, Taylor M, Heginbotham LR, Proctor WR. Long-term increases in excitability in the CA1 region of rat hippocampus induced by β-adrenergic stimulation: possible mediation by cAMP. Journal of Neuroscience. 1992;12:506–517. doi: 10.1523/JNEUROSCI.12-02-00506.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Edwards FA, Konnerth A, Sakmann B, Takahashi T. A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Pflügers Archiv. 1989;414:600–612. doi: 10.1007/BF00580998. [DOI] [PubMed] [Google Scholar]
  20. Goaillard J-M, Legendre P, Vincent P. Serotonin decreases the slow afterhyperpolarization in intralaminar thalamic neurones via 5-HT 7 receptor activation: a study using cAMP imaging and electrophysiological recordings. Society for Neuroscience Abstracts. 2000;26:810.13. [Google Scholar]
  21. Goaillard J-M, Vincent P, Fischmeister R. Simultaneous measurements of intracellular cAMP and L-type Ca2+ current in single frog ventricular myocytes. Journal of Physiology. 2001;530:79–91. doi: 10.1111/j.1469-7793.2001.0079m.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Goldstein SA, Bockenhauer D, O'Kelly I, Zilberberg N. Potassium leak channels and the KCNK family of two-P-domain subunits. Nature Reviews Neuroscience. 2001;2:175–184. doi: 10.1038/35058574. [DOI] [PubMed] [Google Scholar]
  23. Gustafson EL, Durkin MM, Bard JA, Zgombick J, Branchek TA. A receptor autoradiographic and in situ hybridization analysis of the distribution of the 5-HT7 receptor in rat brain. British Journal of Pharmacology. 1996;117:657–666. doi: 10.1111/j.1476-5381.1996.tb15241.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Haas HL, Gähwiler BH. Vasoactive intestinal polypeptide modulates neuronal excitability in hippocampal slices of the rat. Neuroscience. 1992;47:273–277. doi: 10.1016/0306-4522(92)90243-u. [DOI] [PubMed] [Google Scholar]
  25. Haug T, Storm JF. Protein kinase A mediates the modulation of the slow Ca(2+)-dependent K(+) current, I(sAHP), by the neuropeptides CRF, VIP, and CGRP in hippocampal pyramidal neurons. Journal of Neurophysiology. 2000;83:2071–2079. doi: 10.1152/jn.2000.83.4.2071. [DOI] [PubMed] [Google Scholar]
  26. Heidmann DE, Szot P, Kohen R, Hamblin MW. Function and distribution of three rat 5-hydroxytryptamine7 (5-HT7) receptor isoforms produced by alternative splicing. Neuropharmacology. 1998;37:1621–1632. doi: 10.1016/s0028-3908(98)00070-7. [DOI] [PubMed] [Google Scholar]
  27. Jasper JR, Kosaka A, To ZP, Chang DJ, Eglen RM. Cloning, expression and pharmacology of a truncated splice variant of the human 5-HT7 receptor (h5-HT7b) British Journal of Pharmacology. 1997;122:126–132. doi: 10.1038/sj.bjp.0701336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kikuchi C, Nagaso H, Hiranuma T, Koyama M. Tetrahydrobenzindoles: selective antagonists of the 5-HT7 receptor. Journal of Medicinal Chemistry. 1999;42:533–535. doi: 10.1021/jm980519u. [DOI] [PubMed] [Google Scholar]
  29. Lancaster B, Batchelor AM. Novel action of BAPTA series chelators on intrinsic K+ currents in rat hippocampal neurones. Journal of Physiology. 2000;522:231–246. doi: 10.1111/j.1469-7793.2000.t01-1-00231.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lazdunski M. Apamin, a neurotoxin specific for one class of Ca2+-dependent K+ channels. Cell Calcium. 1983;4:421–428. doi: 10.1016/0143-4160(83)90018-0. [DOI] [PubMed] [Google Scholar]
  31. Leidenheimer NJ, Browning MD, Harris RA. GABA A receptor phosphorylation: multiple sites, actions and artifacts. Trends in Pharmacological Sciences. 1991;12:34–87. doi: 10.1016/0165-6147(91)90509-q. [DOI] [PubMed] [Google Scholar]
  32. Lovell PJ, Bromidge SM, Dabbs S, Duckworth DM, Forbes IT, Jennings AJ, King FD, Middlemiss DN, Rahman SK, Saunders DV, Collin LL, Hagan JJ, Riley GJ, Thomas DR. A novel, potent, and selective 5-HT(7) antagonist: (R)-3-(2-(2-(4-methylpiperidin-1-yl) ethyl)pyrrolidine-1-sulfonyl) phenol (SB-269970) Journal of Medicinal Chemistry. 2000;43:342–345. doi: 10.1021/jm991151j. [DOI] [PubMed] [Google Scholar]
  33. McCormick DA, Pape HC. Noradrenergic and serotonergic modulation of a hyperpolarization-activated cation current in thalamic relay neurones. Journal of Physiology. 1990;431:319–342. doi: 10.1113/jphysiol.1990.sp018332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McCormick DA, Prince DA. Noradrenergic modulation of firing pattern in guinea pig and cat thalamic neurons, in vitro. Journal of Neurophysiology. 1988;59:978–996. doi: 10.1152/jn.1988.59.3.978. [DOI] [PubMed] [Google Scholar]
  35. McCormick DA, Williamson A. Modulation of neuronal firing mode in cat and guinea pig LGNd by histamine: possible cellular mechanisms of histaminergic control of arousal. Journal of Neuroscience. 1991;11:3188–3199. doi: 10.1523/JNEUROSCI.11-10-03188.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Matsuoka I, Maldonado R, Defer N, Noël F, Hanoune J, Roques BP. Chronic morphine administration causes region-specific increase of brain type VIII adenylyl cyclase mRNA. European Journal of Pharmacology. 1994;268:215–221. doi: 10.1016/0922-4106(94)90191-0. [DOI] [PubMed] [Google Scholar]
  37. Matsuoka I, Suzuki Y, Defer N, Nakanishi H, Hanoune J. Differential expression of type I, II, and V adenylyl cyclase gene in the postnatal developing rat brain. Journal of Neurochemistry. 1997;68:498–506. doi: 10.1046/j.1471-4159.1997.68020498.x. [DOI] [PubMed] [Google Scholar]
  38. Nicoll RA. The coupling of neurotransmitter receptors to ion channels in the brain. Science. 1988;241:545–551. doi: 10.1126/science.2456612. [DOI] [PubMed] [Google Scholar]
  39. Pape HC. Adenosine promotes burst activity in guinea-pig geniculocortical neurones through two different ionic mechanisms. Journal of Physiology. 1992;447:729–753. doi: 10.1113/jphysiol.1992.sp019026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pedarzani P, Storm JF. PKA mediates the effects of monoamine transmitters on the K+ current underlying the slow spike adaptation in hippocampal neurons. Neuron. 1993;11:1023–1035. doi: 10.1016/0896-6273(93)90216-e. [DOI] [PubMed] [Google Scholar]
  41. Peschanski M, Besson JM. Diencephalic connections of the raphe nuclei of the rat brainstem: an anatomical study with reference to the somatosensory system. Journal of Comparative Neurology. 1984;224:509–534. doi: 10.1002/cne.902240404. [DOI] [PubMed] [Google Scholar]
  42. Pilowsky I, Barrow CG. A controlled study of psychotherapy and amitriptyline used individually and in combination in the treatment of chronic intractable, ‘psychogenic’ pain. Pain. 1990;40:3–19. doi: 10.1016/0304-3959(90)91045-K. [DOI] [PubMed] [Google Scholar]
  43. Price DD. Psychological and neural mechanisms of the affective dimension of pain. Science. 2000;288:1769–1772. doi: 10.1126/science.288.5472.1769. [DOI] [PubMed] [Google Scholar]
  44. Price JL. Thalamus. In: Paxinos G, editor. The Rat Nervous System. San Diego: Academic Press; 1995. pp. 629–648. [Google Scholar]
  45. Qiao JT, Dafny N. Dorsal raphe stimulation modulates nociceptive responses in thalamic parafascicular neurons via an ascending pathway: further studies on ascending pain modulation pathways. Pain. 1988;34:65–74. doi: 10.1016/0304-3959(88)90183-2. [DOI] [PubMed] [Google Scholar]
  46. Sah P. Ca(2+)-activated K+ currents in neurones: types, physiological roles and modulation. Trends in Neurosciences. 1996;19:150–154. doi: 10.1016/s0166-2236(96)80026-9. [DOI] [PubMed] [Google Scholar]
  47. Sah P, Clements JD. Photolytic manipulation of [Ca2+]i reveals slow kinetics of potassium channels underlying the afterhyperpolarization in hippocampal pyramidal neurons. Journal of Neuroscience. 1999;19:3657–3664. doi: 10.1523/JNEUROSCI.19-10-03657.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Shen Y, Monsma FJ, Jr, Metcalf MA, Jose PA, Hamblin MW, Sibley DR. Molecular cloning and expression of a 5-hydroxytryptamine7 serotonin receptor subtype. Journal of Biological Chemistry. 1993;268:18200–18204. [PubMed] [Google Scholar]
  49. Steriade M, Jones EG, McCormick DA. Thalamus. Oxford: Elsevier Science; 1997. Electrophysiological properties of thalamic neurons; pp. 339–392. [Google Scholar]
  50. Thomas DR, Gittins SA, Collin LL, Middlemiss DN, Riley G, Hagan J, Gloger I, Ellis CE, Forbes IT, Brown AM. Functional characterisation of the human cloned 5-HT7 receptor (long form); antagonist profile of SB-258719. British Journal of Pharmacology. 1998;124:1300–1306. doi: 10.1038/sj.bjp.0701946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Vanhoenacker P, Haegeman G, Leysen JE. 5-HT7 receptors: current knowledge and future prospects. Trends in Pharmacological Sciences. 2000;21:70–77. doi: 10.1016/s0165-6147(99)01432-7. [DOI] [PubMed] [Google Scholar]
  52. Vergara C, Latorre R, Marrion NV, Adelman JP. Calcium-activated potassium channels. Current Opinion in Neurobiology. 1998;8:321–329. doi: 10.1016/s0959-4388(98)80056-1. [DOI] [PubMed] [Google Scholar]
  53. Vertes RP, Fortin WJ, Crane AM. Projections of the median raphe nucleus in the rat. Journal of Comparative Neurology. 1999;407:555–582. [PubMed] [Google Scholar]
  54. Vincent P, Brusciano D. Cyclic AMP imaging in neurones in brain slice preparations. Journal of Neuroscience Methods. 2001;108:189–198. doi: 10.1016/s0165-0270(01)00393-4. [DOI] [PubMed] [Google Scholar]
  55. Vizuete ML, Venero JL, Traiffort E, Vargas C, Machado A, Cano J. Expression of 5-HT7 receptor mRNA in rat brain during postnatal development. Neuroscience Letters. 1997;227:53–56. doi: 10.1016/s0304-3940(97)00302-9. [DOI] [PubMed] [Google Scholar]

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