- BB1 receptor
bombesin type 1 receptor
- BB2 receptor
bombesin type 2 receptor
- BB3 receptor
bombesin type 3 receptor
- I GRP
- I H
hyperpolarization-activated inward current
- I T
T-type Ca2+ current
- I–V relationship
inward rectifier K+
paraventricular thalamic nucleus
- R M
canonical transient receptor potential
transient receptor potential vanilloid
- V H
- V M
- • Gastrin-releasing peptide (GRP) is a mammalian bombesin-like peptide that is widely distributed in the CNS. Whereas GRP is known to have a predominant excitatory action on neurons, details of the underlying membrane mechanism remain largely undefined.
- • We investigated GRP-affected receptors and ionic conductances in the midline paraventricular thalamic nucleus, a brain region densely innervated by GRP-like immunoreactive fibres.
- • Perforated patch clamp recording in acute brain slices showed that exposure of paraventricular thalamic nucleus neurons to low nanomolar concentrations of GRP resulted in membrane depolarization with rhythmic burst or tonic firing. These responses were due to a postsynaptic bombesin type 2 receptor-mediated simultaneous suppression of a Ba2+-sensitive inward rectifier K+ conductance and activation of a non-selective cation conductance with transient receptor potential vanilloid 1-like properties.
- • The data provide details on the nature of the receptor and ionic conductances involved in GRP's excitatory influence on midline thalamic neurons.
Abstract Gastrin-releasing peptide (GRP) is a bombesin-like peptide with a widespread distribution in mammalian CNS, where it has a role in food intake, circadian rhythm generation, fear memory, itch sensation and sexual behaviour. While it has been established that GRP predominantly excites neurons, details of the membrane mechanism involved in this action remain largely undefined. We used perforated patch clamp recording in acute brain slice preparations to investigate GRP-affected receptors and ionic conductances in neurons of the rat paraventricular thalamic nucleus (PVT). PVT is a component of the midline and intralaminar thalamus that participates in arousal, motivational drives and stress responses, and exhibits a prominence of GRP-like immunoreactive fibres. Exposure of PVT neurons to low nanomolar concentrations of GRP induced sustained TTX-resistant membrane depolarizations that could trigger rhythmic burst discharges or tonic firing. Membrane current analyses in voltage clamp revealed an underlying postsynaptic bombesin type 2 receptor-mediated inward current that resulted from the simultaneous suppression of a Ba2+-sensitive inward rectifier K+ conductance and activation of a non-selective cation conductance with biophysical and pharmacological properties reminiscent of transient receptor potential vanilloid (TRPV) 1. A role for a TRPV1-like conductance was further implied by a significant suppressant influence of a TRPV1 antagonist on GRP-induced membrane depolarization and rhythmic burst or tonic firing. The results provide a detailed picture of the receptor and ionic conductances that are involved in GRP's excitatory action in midline thalamus.
Gastrin-releasing peptide (GRP), a 27-amino acid peptide, and the decapeptide neuromedin B (NMB) are mammalian analogues of the family of bombesin (BB) and BB-like peptides originally described in amphibians (Ohki-Hamazaki, 2000; Jensen et al. 2008). GRP and NMB exert their biological actions through an interaction with GRP preferring or BB type 2 (BB2) and NMB preferring or bombesin type 1 (BB1) receptors, which are both coupled to Gq/G11 proteins to modulate phospholipase C (PLC)-regulated second messenger pathways (Jensen et al. 2008). GRP, NMB and their receptors are present in the CNS, with distributions in the forebrain, brainstem and spinal cord (Zoeller et al. 1989; Wada et al. 1990, 1992; Mikkelsen et al. 1991; Ladenheim et al. 1992; Moody & Merali, 2004; Sun & Chen, 2007; Sakamoto et al. 2008).
Central GRP and its receptor have been implicated in several aspects of physiology and behaviour, most notably food intake, circadian rhythm generation, fear-related memory processing, itch sensation and sexual behaviour (McArthur et al. 2000; Ladenheim et al. 2002; Shumyatsky et al. 2002; Moody & Merali, 2004; Sun & Chen, 2007; Sakamoto et al. 2008). While it has been established that GRP may exert its central role through a predominantly excitatory influence on neurons, details of the receptors and ionic conductances underlying this action are still largely unknown. To date, GRP and BB have been suggested to excite mammalian neurons through suppression of an unidentified hyperpolarizing K+ conductance, activation of a largely uncharacterized depolarizing Na+ permeable conductance, activation of a Na+/Ca2+ exchanger, and modulation of a fast delayed rectifier K+ conductance (Pinnock & Woodruff, 1991; Reynolds & Pinnock, 1997; Lee et al. 1999; Van den Pol et al. 2009; Gamble et al. 2011). When simultaneously investigated, the mediating receptor was BB2-like (Lee et al. 1999).
Among several brain regions reported to be innervated by GRP-containing fibres is the paraventricular thalamic nucleus (PVT), a midline thalamic cell group that is integral to the limbic system and deemed to participate in arousal and awareness, motivational drives and stress responses (Mikkelsen et al. 1991; Van der Werf et al. 2002; Sewards & Sewards, 2003; Price & Drevets, 2010). Upon re-examination, we noted a dense GRP-like immunoreactive labelling of fibres in select midline and intralaminar thalamic nuclei, including PVT. We subsequently used perforated patch clamp recording in acute in vitro brain slices to evaluate the membrane mechanism in PVT neurons influenced by exposure to exogenous GRP. The results imply that GRP, via an interaction with postsynaptic BB2 receptors, excites PVT neurons through suppression of Ba2+-sensitive inward rectifier K+ (Kir) conductance and concomitant activation of a non-selective cation conductance with a transient receptor potential vanilloid (TRPV)-1-like profile.
Animals and ethical approval
Experiments used male Wistar rats (age 21–50 days) bred in-house and maintained on a 12 h light/12 h dark cycle (lights on at 06.00 h). Experimental protocols conformed to the Canadian Council for Animal Care guidelines and were approved by the Ottawa Hospital Research Institute Animal Care and Use Committee.
For immunohistochemistry of GRP, four rats (age 35–50 days) were deeply anaesthetized with intraperitoneal sodium pentobarbital (50 mg kg−1) and perfused transcardially with 4% paraformaldehyde in 0.1 m PBS (pH 7.4). The brains were stored in this fixative for 1–2 days at 4°C and subsequently kept in 0.1 m PBS (pH 7.4) at 4°C until sectioning. Coronal 30–50 μm sections were made using a vibrating blade microtome (Leica VT1000S; Leica Microsystems, Richmond Hill, ON, Canada). Labelling of GRP was obtained with a primary rabbit polyclonal antibody (ab222632; Abcam, Cambridge, MA, USA: dilution 1:5000) following an incubation procedure described previously (Hermes et al. 2009). A control experiment consisted of absorbing the antibody working dilution with 30 nm GRP 3 h before and during overnight incubation of the brain sections, which resulted in complete elimination of labelling.
Preparation of thalamic slices for electrophysiology
To minimize inadvertent effects by anaesthetics, rats (age 21–40 days) were killed by decapitation using a guillotine, between 08.00 and 11.00 h. Their brains were gently removed from the skull and immersed in an ice-cold, oxygenated (95% O2, 5% CO2), sucrose-based solution of the following composition (in mm): sucrose 200, KCl 2.5, NaH2PO4 1.2, NaHCO3 26, d-glucose 10, CaCl2 1 and MgCl2 6. Brains were sliced at 400 μm using a vibrating blade microtome (Leica VT1000S). Slices were subsequently maintained in oxygenated ACSF of the following composition (in mm): NaCl 120, KCl 3, NaH2PO4 1.2, NaHCO3 26, d-glucose 10, CaCl2 2.4 and MgCl2 1.2, (pH 7.3, osmolality 295–300 mosmol kg−1), first for 30–45 min at 30°C and then for >30 min at room temperature. For patch clamp recording, slices were submerged in a custom-built recording chamber and perfused with oxygenated ACSF at 32–34°C, at a flow rate of 3–4 ml min−1.
Patch clamp recording
Cell attachment was accomplished through a ‘blind’ approach and using patch pipettes pulled from thin-walled borosilicate glass capillaries (Sutter Instruments, Novato, CA, USA) with a Flaming-Brown P-97 horizontal puller (Sutter Instruments). Whole cell voltages and currents were obtained using a modified perforated patch recording method employing both amphotericin B and gramicidin as perforating substances: full perforation of the patched membrane was usually achieved 15–20 min after cell attachment. The composition of the pipette solution was (in mm): potassium gluconate 135, KCl 10, Hepes 10, EGTA 1 (pH 7.4 with 2 mm KOH and 4 mm NaOH), to which amphotericin B (70 μg ml−1) and gramicidin (30 μg ml−1) were added from freshly prepared 10 mg ml−1 DMSO solutions. To enable cell attachment, tips of the patch pipettes were immersed in amphotericin B- and gramicidin-free pipette solution and filled by applying negative pressure to the back opening. For recording of non-selective cation currents, caesium gluconate or caesium methanesulphonate and CsCl (pH 7.4 with CsOH and 4 mm NaOH) replaced potassium gluconate, KCl and KOH: with the caesium methanesulphonate-based pipette solution gramicidin was reduced to 5 μg ml−1. A gradual membrane depolarization and broadening of action potentials or low-threshold Ca2+ potentials indicated the feasibility of internal Cs+ block of K+ channels in the perforated patch configuration. Filled pipettes had an open resistance of 6–8 MΩ.
Series resistance of recordings ranged between 14 and 30 MΩ, was compensated for >50%, and regularly checked by monitoring current responses to brief 5 mV hyperpolarizing steps. Recordings were terminated when the series resistance (after full perforation) changed by more than ∼15%. Electrical signals were amplified with an Axopatch 200B amplifier (MDS Analytical Technologies, Sunnyvale, CA, USA) and stored using a Digidata 1200 analog-to-digital converter (MDS Analytical Technologies) and Clampex 9.2 and Axoscope 9.2 software (part of pClamp; MDS Analytical Technologies). Data were filtered at 1 kHz and sampled at either 5 kHz (Clampex) or 1 kHz (Axoscope).
Neurons were recorded in midline PVT, intralaminar central medial thalamic nucleus and mediodorsal thalamic nucleus, from bregma −1.30 mm to bregma −3.30 mm (Paxinos & Watson, 1998), and at zeitgeber time 5–17 (zeitgeber time is in hours: 0 is lights on in the original light/dark cycle). In current clamp recordings (without injection of holding currents), following registration of basic intrinsic properties of PVT neurons that included membrane potential (VM) at rest, frequency of spontaneous action potential discharge and input resistance (RM: by subjecting the cell to 20 pA negative current pulses), the influence of bath application of drugs on VM and action potential discharge was analysed. In voltage clamp recordings the influence of bath application of drugs on membrane currents in PVT neurons, clamped at a holding potential (VH) of –70 mV, was analysed. To obtain details of these membrane currents, cells were also subjected to either a protocol that stepped VH from −60 to –130 mV in 10 mV steps of a duration of ≥2.4 s, or a protocol that ramped VH from +20 to –130 mV, at a rate of 10 mV s−1.
Data analysis and statistics
Data collected with Axoscope and Clampex were analysed using Clampfit 9.2 software (part of pClamp; MDS Analytical Technologies). Drug-induced changes in VM were assessed by averaging 10–20 s of data. Membrane depolarizations were deemed significant and indicating responsiveness to GRP when exceeding 3 mV. Patterns of action potential discharge were analysed from periods of 3 to 5 min in cases of rhythmic burst firing and from periods of 1 to 2 min in cases of tonic firing. Drug-induced changes in membrane currents were determined at VH−70 mV and, where step protocols were used, at VH−60 to –130 mV by subtracting voltage step-induced membrane currents in control from those in drug or vice versa. Currents were measured both at 40–60 ms following the start of voltage steps and at 50–70 ms before the end of voltage steps. In addition, the amplitude of the T-type Ca2+ current (IT), peaking ∼20–25 ms after termination of voltage steps to VH≤−90 mV, was measured. Resting membrane conductance was calculated from the current deflections 40–60 ms following the start of a voltage step to VH−90 mV. In cases where ramp protocols were used, drug-induced changes in membrane currents were obtained by subtracting voltage ramp-induced membrane currents in control from those in drug or vice versa: the resulting data were reduced by a factor 100 and imported in GraphPad Prism 4.03 software (GraphPad Software, La Jolla, CA, USA). Here, drug-induced maximum inward currents were visually identified and measured by averaging data points ±5 mV from peak. Reversal potentials and slopes were determined by linear regression of data points within a chosen voltage range. The Cs+/Na+ permeability ratio of the GRP-activated current was calculated from the reversal potential in the nominally zero extracellular Ca2+ and using a standard formula (Vennekens et al. 2000). The Ca2+/Na+ permeability ratio was calculated from the reversal potential in control, Ca2+-containing, ACSF and the Cs+/Na+ permeability ratio (Vennekens et al. 2000).
Voltage measures in current clamp data were corrected for a liquid junction potential of 15.8 mV, calculated with the liquid junction potential module of Clampex 9.2 software. Voltage commands in voltage clamp recordings were corrected for 20 mV to enable comparison with data obtained in ACSF containing Tris-Cl instead of NaCl, which needed correction for a liquid junction potential of 21 mV. All data are expressed as mean ±s.e.m. The two-tailed Student's paired or unpaired t test was used for within-cell and between-cell comparisons, respectively. For non-normally distributed data, determined with the Kolmogorov–Smirnov normality test, the Wilcoxon signed-rank test or two-tailed Mann–Whitney U test was used for within-cell and between-cell comparisons, respectively. Differences were considered statistically significant at P < 0.05.
GRP was purchased from American Peptide (Sunnyvale, CA, USA), Phoenix Pharmaceuticals (Burlingame, CA, USA) or Tocris Bioscience (Ellisville, MO, USA). TTX was purchased from Ascent Scientific (Princeton, NJ, USA). 2,3-Dioxo-6-nitro-1,2,3,4-tretahydrobenzo(f)quinoxaline-7-sulphonamide disodium salt (NBQX), d-(–)-2-amino-5-phosphonopentanoic acid (d-AP5), bicuculline meth-ochloride (BIC), 6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid hydrobromide (gabazine), (S)-N-[[1-(5-methoxy-2-pyridinyl)cyclohexyl]methyl]-α-methyl-α-[[[-(4-nitrophenyl)amino]carbonyl]amino]-1H-indole-3-propanamide (PD 176252), (S)-α-methyl-α-[[[(4-nitrophenyl)amino]carbonyl]amino]-N-[[1-(2-pyridinyl)cyclohexyl]methyl]-1H-indole-3-propanamide (PD 168368), 4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride (ZD 7288), (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH 23390), capsazepine (CPZ), 4′-chloro-3-methoxycinnamanilide (SB 366791) and capsaicin were purchased from Tocris Bioscience. Amphotericin B, gramicidin and [d-Tpi6, Leu13,Ψ(CH2-NH),Leu14]bombesin(6-14) (RC-3095) were purchased from Sigma-Aldrich (Oakville, ON, Canada). [d-Phe6,Des-Met14]Bombesin(6-14)ethylamide ([d-Phe6,Des-Met14]Bn(6-14)EA) was purchased from American Peptide. 4-Methyl-2-(piperidin-1-yl)quinoline (ML204) was kindly provided by Julie Le Engers of Vanderbilt Medical Center (Nashville, TN, USA).
Stock solutions (usually 1000×, for freezing) of GRP, TTX, NBQX, d-AP5, BIC, gabazine, [d-Phe6,Des-Met14]Bn(6-14)EA, ZD 7288, SCH 23390 and ML204 were made in distilled water. Stock solutions of PD 176252, CPZ, SB 366791, capsaicin and RC-3095 were made in DMSO. Stock solution of PD 168368 was 100× and prepared in a 45% solution of (2-hydroxypropyl)-β-cyclodextrin (Sigma). All drugs were bath applied.
Gastrin-releasing peptide-like immunoreactive fibres in midline and intralaminar thalamus
The presence of GRP-like immunoreactive material in the thalamus was limited to midline and intralaminar nuclei. Most notable was a high density of labelled fibres in midline PVT at posterior levels where they clearly outlined the borders of the nucleus (Fig. 1B). Similar intense fibre labelling was seen in select subdivisions of the intralaminar thalamus, in particular the central medial, paracentral and central lateral thalamic nuclei (Fig. 1A and B). A moderate density of labelled fibres was present in the midline PVT at anterior levels (Fig. 1A). Modest labelling was also seen in the intermediodorsal, rhomboid and reunions thalamic nuclei while few fibres were observed in the caudal intralaminar parafascicular thalamic nucleus (not shown). The paratenial and mediodorsal thalamic nuclei were devoid of labelled fibres (Fig. 1A and B). No somas displaying GRP-like immunoreactivity were detected.
Gastrin-releasing peptide induces membrane depolarization and low frequency rhythmic burst or tonic firing
Initial investigations focused on the influence of GRP on PVT neuronal excitability. We detected that 75 s bath applications of GRP induced minimal and maximal membrane depolarizations within a narrow low nanomolar concentration range: 2.1 ± 1.4 mV with 2 nm (n= 3); 3.8 ± 0.6 mV with 5 nm (n= 3); 8.0 ± 3.3 mV with 10 nm (n= 3); 11.1 ± 0.8 mV with 20 nm (n= 50); and 11.5 ± 2.2 mV with 50 nm (n= 6). As 20 nm resulted in a maximum response, we chose this concentration for all further experiments detailed below.
In the group of cells tested with 20 nm GRP 4/50 PVT neurons were unresponsive. The 46 responsive neurons showed membrane depolarizations (range 3.1–23.2 mV) that resulted in three distinct action potential responses:
- 1Low frequency (0.02–0.33 Hz) rhythmic burst firing, resulting from recurring 2–16 s long series of 2–13 high frequency (33–200 Hz) bursts of action potentials interrupted by 3–50 s long silent periods (Fig. 2A). Such responses lasted 6–32 min and were seen in 20 neurons. These cells had a resting VM of −84.2 ± 0.8 mV, an RM of 592 ± 40 MΩ and were silent (n= 14) or showed rare single bursts of action potentials (n= 6) before GRP application.
- 2Tonic firing with a mean frequency of 4.9 ± 0.4 Hz (Fig. 2B). Such responses persisted for 6–30 min and occurred in 18 neurons. These cells had a resting VM of −77.7 ± 1.2 mV, an RM of 665 ± 57 MΩ, and were silent (n= 8), showed rare single bursts of action potentials (n= 5), or displayed low frequency rhythmic burst firing (n= 5) before GRP application.
- 3No initiation of firing or a cessation of action potential discharges were observed in eight neurons (not shown). These cells had a resting VM of −77.7 ± 2.8 mV, an RM of 630 ± 54 MΩ, and were silent (n= 4), showed rare single bursts of action potentials (n= 2), or displayed low frequency rhythmic burst firing (n= 2) before GRP application.
The type of action potential response was not related to the neuron's rostrocaudal location in PVT or the zeitgeber time of recording. In addition, low frequency rhythmic burst and tonic firing responses were not associated with differences in the amplitude of the GRP-induced membrane depolarization; however, silent responses were linked to a significantly smaller depolarization (Fig. 2C). Notably, tonic firing responses were associated with a significantly more depolarized VM in GRP, consequent to a more depolarized resting VM (Fig. 2D). Further evidence that the level of resting VM may be an important factor in determining the type of action potential response was obtained in experiments employing two consecutive GRP applications: here tonic firing following a first GRP application could be transformed into rhythmic bursting following a second GRP application when VM was maintained more hyperpolarized by constant negative current injection (n= 3) (not shown). The data are consistent with observations in thalamic relay neurons; in these neurons the level of VM and the occurrence of burst or tonic firing is in part determined by two-pore domain K+ channels (Bal & McCormick, 1993; Meuth et al. 2003).
For comparison, we also sampled neurons in the central medial thalamic nucleus where the density of GRP-like immunoreactive fibres was comparable to that in PVT (Fig. 1). Here, four of seven cells responded to GRP with a reversible 13.5 ± 2.7 mV membrane depolarization resulting in rhythmic burst firing in three cells and tonic firing in the other (not shown). By contrast, in the mediodorsal thalamic nucleus, a site that lacked GRP-like immunoreactive fibres, none of five neurons tested showed any membrane response to GRP applied at concentrations up to 200 nm (not shown).
Gastrin-releasing peptide's membrane influence is mediated by postsynaptic bombesin type 2 receptors
To distinguish between a pre- or postsynaptic location of action, we examined GRP's influence on PVT neurons in ACSF containing TTX (1 μm), NBQX (2 μm), d-AP5 (10 μm) and BIC (10 μm) or gabazine (10 μm). In these conditions GRP induced a reversible membrane depolarization of 13.8 ± 2.8 mV (n= 5), not significantly different from that in control ACSF (P= 0.20, Student's unpaired t test), implying a postsynaptic site of action (data not shown).
GRP has been reported to interact preferentially with BB2 receptors (Jensen et al. 2008). Interestingly, of the BB receptor family it is only the BB1 receptors (which exhibit submicromolar affinity for GRP) and bombesin type 3 (BB3) receptors (which do not bind GRP) that have been detected in the region of PVT (Ladenheim et al. 1992; Wada et al. 1992; Jennings et al. 2003). To assess the nature of the receptor mediating GRP's postsynaptic action, we next used recordings in voltage clamp mode to test the influence of several BB receptor antagonists. We first noted that PVT neurons, clamped at VH–70 mV in ACSF containing TTX, NBQX, d-AP5 and BIC or gabazine (these blockers were used in all subsequent voltage clamp experiments), responded to a continuous (to allow for equilibration of peptide concentration in the recording chamber) bath application of GRP with a sustained inward current (IGRP) of −47.4 ± 4.7 pA (n= 12) (range −26.4 to −85.8 pA) that showed no evidence of desensitization (Fig. 3A and B). In 10 μm of the non-selective BB1/BB2 receptor antagonist PD 176252 (Jensen et al. 2008: applied for ≥10 min before and during GRP) IGRP was reduced to 14% of control (n= 5; P < 0.01, Mann–Whitney U test) (Fig. 3B). By contrast, in 2 μm of the selective BB1 receptor antagonist PD 16368 (Jensen et al. 2008) IGRP was not different from control (n= 6; P= 0.28, Mann–Whitney U test) (Fig. 3B). These data suggest a predominant involvement of BB2 receptors.
To obtain further evidence in favour of a role for BB2 receptors, we also tested two selective BB2 receptor antagonists: [d-Phe6,Des-Met14]-Bn-(6-14)-EA and RC-3095 (Jensen et al. 2008). One or 3 μm[d-Phe6,Des-Met14]-Bn-(6-14)-EA has previously been shown to block GRP membrane actions in the hippocampus (Lee et al. 1999) and amygdala (Shumyatsky et al. 2002). In 1 μm[d-Phe6,Des-Met14]-Bn-(6-14)-EA IGRP was almost completely blocked (n= 4; P < 0.01, Mann–Whitney U test) (Fig. 3B). In addition, in 2 μm RC-3095 IGRP was reduced to 24% of control (n= 5; P < 0.01, Mann–Whitney U test) (Fig. 3B). Collectively, these data support the notion that GRP's postsynaptic action on PVT neurons is mediated primarily by BB2 receptors.
Gastrin-releasing peptide generates a voltage-dependent inward current
To obtain further details of IGRP, PVT neurons that were exposed to a continuous GRP application were also subjected to a repeating protocol that stepped VH from −60 to –130 mV in 10 mV steps (Fig. 4A). Repeated execution of the step protocol showed that control membrane current responses remained stable over periods exceeding 20 min. Subtraction of voltage step-induced control membrane current responses from those in GRP (usually after 8–10 min of continuous GRP application) revealed that IGRP had a non-linear relationship to VH in the membrane potential range tested: the amplitude of IGRP decreased linearly from −58.8 ± 5.8 pA at VH–60 mV (n= 12) to −29.0 ± 5.4 pA at VH–90 mV (n= 12), but changed little negative to VH–90 mV (i.e. −23.2 ± 7.5 pA at VH–130 mV, n= 12) and showed no reversal of polarity (Fig. 4B).
Analysis of other aspects of the current response to voltage steps showed that the amplitude of a hyperpolarization-activated inward current (IH), activated with hyperpolarizing steps to VH≤−100 mV (with a maximum value of −76.6 ± 8.3 pA with a step to VH–120 mV, n= 12), was not affected by GRP (P > 0.05 for all steps to VH≤−100 mV, Wilcoxon signed-rank test) (not shown). These data indicate that IGRP is not due to an enhanced activation of IH. In addition, the amplitude of IT, activated following hyperpolarizing steps to VH≤−90 mV (with a maximum value of −541.2 ± 53.6 pA following a step to VH–100 mV, n= 12), was not changed by GRP (P > 0.05 for all steps to VH≤−90 mV, Student's paired t test) (not shown).
The current–voltage relationship (I–V relationship) of IGRP could reflect suppression of a K+ conductance as extrapolation of the linear segment between VH−60 and –90 mV reverses polarity near the K+ equilibrium potential; the lack of an actual reversal potential could arise because of an inadequate voltage clamp at negative VHs. It may also represent simultaneous alterations in K+ (suppression) and cation (activation) conductances (Shen & North, 1992), or activation of a voltage-gated non-selective cation conductance with a negative slope at membrane potentials negative to ∼−30 mV (Haj-Dahmane & Andrade, 1996). To assess the contribution of K+ and non-selective cation conductances to IGRP, we next analysed the effect of GRP in ACSF containing 27.2 mm Na+ instead of 147.2 mm Na+ (by replacing 120 mm NaCl with equimolar Tris-Cl: low Na+ ACSF), a procedure known to attenuate transmitter-generated inward currents mediated by non-selective cation conductances (cf. Haj-Dahmane & Andrade, 1996).
Suppression of a Ba2+-sensitive inward rectifier K+ conductance contributes to gastrin-releasing peptide-generated current
PVT neurons recorded in low Na+ ACSF and 50 μm ZD 7288 (to block residual IH) showed inwardly rectifying membrane currents reminiscent of currents mediated by Kir2 channels (Kubo et al. 1993). Continuous bath application of GRP in low Na+ ACSF generated a sustained inward current of −12.3 ± 1.5 pA at VH–70 mV (n= 8) and resulted in an apparent reduction of maximum inward currents with steps to VH–130 mV (Fig. 5A). Subtraction of voltage step-induced membrane currents in GRP from those in control revealed suppression of a membrane current that reversed polarity at −104.6 ± 2.7 mV, near the calculated K+ equilibrium potential of −103.2 mV, and had inwardly rectifying properties with a maximum suppressed current of −44.0 ± 2.6 pA at VH−130 mV (n= 8) (Fig. 5B and C). These data indicate that suppression of a Kir conductance contributes to IGRP.
Kir conductances can be mediated by channels from seven subfamilies (Hibino et al. 2010). Three ion channel subfamilies have an established role in regulation of neuronal excitability: Kir2, which form open or constitutively active Kir channels; Kir3, which form G protein-coupled Kir channels that are usually closed but in certain conditions can be open due to tonic activation by a Gi/Go protein-coupled receptor; Kir6, which form ATP-sensitive Kir channels in complexes with sulphonylurea receptors and are open when ATP levels are low (Stanfield et al. 2002; Hibino et al. 2010). In theory, all three Kir channel types could be modulated by GRP through BB2 receptor-mediated activation of PLC; PLC cleaves the membrane phosphoinositide phosphatidylinositol 4,5-biphosphate, which is required for their normal physiological properties (Stanfield et al. 2002; Jensen et al. 2008; Hibino et al. 2010). Kir3 channels could also be modulated through PLC-mediated activation of protein kinase C (Stevens et al. 1999), or by a direct interaction of Gq/G11 protein α subunits with the channel (Kawano et al. 2007). Kir2 and Kir6, but not Kir3, subfamily members have been detected in PVT (Horio et al. 1996; Thomzig et al. 2005; Saenz del Burgo et al. 2008). To assess the involvement of Kir2, Kir3 and Kir6 channels, we next tested the influence of several selective blockers/antagonists on the maximum GRP-suppressed current at VH–130 mV. In 30 μm Ba2+ (applied for ≥10 min before and during GRP), which preferentially blocks Kir2 channels (Lesage et al. 1995; Kung et al. 2008; Hibino et al. 2010), inwardly rectifying membrane currents in PVT neurons were absent (not shown) and the GRP-suppressed current abolished (n= 4; P < 0.01, Mann–Whitney U test) (Fig. 5C). In SCH 23390 (20–100 μm), a selective blocker of activated Kir3.1 and 3.2 channels (Kuzhikandathil & Oxford, 2002), the GRP-suppressed current was not different from control (n= 4; P= 0.81, Mann–Whitney U test) (Fig. 5C). Also in tolbutamide (200 μm), a selective antagonist of Kir6 channel–sulphonylurea receptor complexes (Hibino et al. 2010), the GRP-suppressed current was unchanged (n= 4; P= 0.93, Mann–Whitney U test) (Fig. 5C). Taken together, these data suggest that GRP suppresses a Ba2+-sensitive, possibly type 2, Kir conductance.
The reduced amplitude of inward currents in low Na+ ACSF suggests an additional contribution to IGRP, presumably by GRP activation of non-selective cation conductance. We next investigated biophysical and pharmacological aspects of this conductance.
Activation of a transient receptor potential vanilloid 1-like conductance contributes to gastrin-releasing peptide-generated current
Voltage-dependent properties of a GRP-activated non-selective cation conductance were investigated using a protocol that ramped VH from +20 to –130 mV (Fig. 6A). Neurons were clamped at VH–70 mV and recorded in ACSF that contained Ba2+ to exclude GRP suppression of the Kir conductance. To improve voltage clamp, we also replaced KCl with CsCl in the ACSF to block activation of IH, and K+ with Cs+ in the pipette solution to block voltage-activated K+ currents. Cs+ has been reported to have no appreciable effect on neurotransmitter activation of non-selective cation conductances (cf. Haj-Dahmane & Andrade, 1996). Continuous bath application of GRP in these recording conditions resulted in a sustained inward current of −23.4 ± 6.8 pA at VH–70 mV (n= 7). Subtraction of voltage ramp-induced membrane current responses in control from those in GRP (Fig. 6A) revealed GRP activation of a non-selective cation current with the following features:
- 1) A linear relationship to VH between +20 and –40 mV, with a positive slope of 1.60 ± 0.01 (n= 7) (Fig. 6B).
- 2) A reversal potential of +1.8 ± 4.8 mV (n= 7) (Fig. 6B and C).
- 3) A maximum inward current of −59.6 ± 5.2 pA at VH−38.2 ± 1.6 mV (n= 7) (Fig. 6B and D).
- 4) A progressive reduction of inward current at membrane potentials negative to VH–40 mV, resulting in a negative slope of −0.83 ± 0.01 between VH–40 and –90 mV (n= 7) (Fig. 6B).
We subsequently estimated ion permeability ratios by measuring reversal potentials in ACSF with different ionic compositions. In nominally Ca2+-free ACSF (by replacing CaCl2 with equimolar NaCl) the reversal potential was −12.9 ± 3.5 mV (n= 7) (Fig. 6C), enabling the calculation of a Cs+/Na+ permeability ratio of 1.5 (Vennekens et al. 2000). In nominally Ca2+-free/low Na+ ACSF the reversal potential was −45.9 ± 2.8 mV (n= 5) (Fig. 6C), similarly yielding a Cs+/Na+ permeability ratio near 1.0 (1.1). From the Cs+/Na+ permeability ratio (1.5) and the reversal potential in control, Ca2+-containing, ACSF (+1.8 mV) a Ca2+/Na+ permeability ratio of 22.3 could be determined (Vennekens et al. 2000). These findings indicate that the GRP-activated non-selective cation conductance has a relative permeability of Ca2+ >Cs+≈Na+ (Fig. 6C).
We also noted that the GRP-activated current in nominally Ca2+-free ACSF had a much larger amplitude, reflected by a maximum inward current of −193.9 ± 29.5 pA or 325% of that in control ACSF (n= 7; P < 0.01, Student's unpaired t test) (Fig. 6D). In addition, in nominally Ca2+-free/low Na+ ACSF the maximum inward current was −11.0 ± 4.9 pA or 19% of that in control ACSF (n= 5; P < 0.001, Student's unpaired t test) (Fig. 6D), demonstrating that Na+ is the predominant charge carrier of the inward current. Taken together, these findings indicate that activation of a non-selective cation conductance with selective Ca2+ permeability and characteristic voltage- and extracellular Ca2+-dependent properties contributes to IGRP.
Neurotransmitter-activated non-selective cation currents with similar characteristics described previously in rat native brain tissue have been suggested to involve ion channels of the transient receptor potential family (e.g. Faber et al. 2006; Meis et al. 2007). Indeed, the I–V relationship of the GRP-activated current resembles that of currents carried by canonical transient receptor potential (TRPC)-4 and -5 channels that form heteromers with TRPC1, or homomeric TRPV1 channels (Gunthorpe et al. 2000; Strübing et al. 2001; Clapham, 2003; Wu et al. 2010). The selective Ca2+ permeability and characteristic sensitivity to extracellular Ca2+ are features of TRPV subfamily members, including TRPV1 (Schaefer et al. 2000; Xu et al. 2002; Clapham, 2003; Samways & Egan, 2011). TRPC5 and TRPV1, but not TRPC4, have been detected in midline and intralaminar thalamus (Mezey et al. 2000; Roberts et al. 2004; Fowler et al. 2007). To assess the involvement of TRPC4, TRPC5 and TRPV1, we next tested several compounds for their effectiveness in potentiating/reducing the maximum GRP-activated inward current. In La3+ (100 μm), which typically potentiates activation of TRPC4 and -5 or their heteromeric configurations with TRPC1 (Schaefer et al. 2000; Strübing et al. 2001), the GRP-activated current was reduced to 62% of control (n= 5; P < 0.05, Student's unpaired t test) (Fig. 6D). In ML204 (20–100 μm), a novel selective antagonist of TRPC4 and -5 channels (Miller et al. 2011), the GRP-activated current was unchanged (n= 5; P= 0.83, Student's unpaired t test) (Fig. 6D). These data suggest that TRPC4 or -5 channels do not underlie the non-selective cation conductance. We subsequently evaluated the effect of two TRPV1 competitive antagonists, CPZ (20 μm) and SB 366791 (10 μm) (Caterina & Julius, 2001; Gunthorpe et al. 2004). In CPZ the GRP-activated current was reduced to 59% of control (n= 5; P < 0.05, Student's unpaired t test) (Fig. 6D). SB 366791 was more potent and resulted in a reduction to 35% of control (n= 5; P < 0.01, Student's unpaired t test) (Fig. 6D). These results imply that the GRP-activated non-selective cation conductance has TRPV1-like pharmacological properties. As TRPV1 is not La3+ sensitive (Clapham, 2007), our observations also raise the possibility of a heteromeric channel structure that includes subunits other than TRPV1. These may include La3+-sensitive TRPV2 and TRPV3 (Smith et al. 2002; Liapi & Wood, 2005; Rutter et al. 2005; Clapham, 2007) or, in the case of cross subfamily heteromerization, La3+-sensitive TRPC1 (Clapham, 2007; Ma et al. 2011).
Capsaicin and gastrin-releasing peptide activate the same membrane conductance
The presence of TRPV1 in midline and intralaminar thalamus has been a topic of controversy (Mezey et al. 2000; Roberts et al. 2004; Cavanaugh et al. 2011). Therefore, we sought to confirm the existence of functional TRPV1 channels in PVT neurons by assessing the influence of capsaicin, the prototypical TRPV1 agonist (Caterina & Julius, 2001). Continuous bath application of 10 μm capsaicin generated a membrane current in PVT neurons that showed I–V characteristics similar to that of the GRP-activated non-selective cation current. Thus, subtraction of voltage ramp-induced membrane current responses in control from those obtained in capsaicin revealed a capsaicin-activated current with a linear relationship to VH between +20 and –40 mV and a positive slope of 1.36 ± 0.01 (n= 7; P > 0.05 vs. GRP), reversal potential of −1.8 ± 5.5 mV (n= 7; P= 0.63 vs. GRP, Student's unpaired t test), maximum inward current of −51.9 ± 6.0 pA at VH−39.3 ± 2.8 mV (n= 7; P= 0.35 vs. GRP, Student's unpaired t test), and negative slope of −0.91 ± 0.02 between VH−40 and –90 mV (n= 7; P > 0.05 vs. GRP) (Fig. 7A). In contrast to GRP, the capsaicin-activated current decreased in amplitude after obtaining a maximum value 3–4 min into the capsaicin application (to 67% of maximum values after 8 min of capsaicin administration), presumably due to desensitization (Caterina & Julius, 2001). Although potentially through separate mechanisms in view of the different desensitization properties, these data suggest that GRP and capsaicin activate the same membrane conductance. It might therefore be expected that one compound would occlude the effect of the other. Indeed, the maximum inward current generated by simultaneous GRP and capsaicin activation (achieved through a 8–10 min bath application of GRP followed by a 3–4 min bath application of GRP plus capsaicin) was −63.4 ± 9.6 pA (n= 4), not significantly different from either the GRP- or the capsaicin-activated maximum inward current (P= 0.79 and 0.41, respectively, Mann–Whitney U test) (Fig. 7B). These results validate the notion that GRP activates a TRPV1-like conductance in PVT neurons.
Transient receptor potential vanilloid 1 antagonism reduces gastrin-releasing peptide-induced membrane depolarization and rhythmic burst or tonic firing
As our voltage clamp data do not provide information about somatic and dendritic distribution, or about functional interactions with other conductances, the extent to which activation of the TRPV1-like conductance contributes to GRP-induced excitability changes in PVT neurons remains unclear. Indeed, suppression of the Ba2+-sensitive Kir conductance alone can be sufficient for membrane depolarization and initiation of action potential discharge (Stanfield et al. 2002; Hibino et al. 2010). To get an insight into the role of the TRPV1-like conductance, we next assessed the effect of TRPV1 antagonism on GRP-induced rhythmic burst and tonic firing. The TRPV1 antagonist SB 366791 (10 μm) is a suitable tool as it had no effect on intrinsic membrane properties of PVT neurons, including resting membrane conductance (n= 6; P= 0.15 vs. control, unpaired Student's t test), IH (n= 6; P= 0.24 vs. control, Mann–Whitney U test) and IT (n= 6; P= 0.21 vs. control, unpaired Student's t test) (not shown), consistent with an earlier report (Gunthorpe et al. 2004).
We employed two consecutive bath applications of 20 nm GRP (for 75 s, at an interval of 20–25 min) to allow identification of the type of action potential response following the first application. The second GRP administration in control experiments generated a response identical to the first, with similar membrane depolarizations (13.6 ± 1.7 mV vs. 13.2 ± 1.7 mV, n= 9; P= 0.58, paired Student's t test) and action potential responses (44% rhythmic bursting, 45% tonic firing and 11% silent with both applications) (data not shown). In contrast, a second GRP application in the presence of 10 μm SB 366791 gave rise to membrane depolarizations that were reduced by 60% (5.3 ± 1.2 mV vs. 13.2 ± 1.2 mV, n= 13; P < 0.001, paired Student's t test) (Fig. 8A and B). Moreover, with GRP in SB 366791 most (69%) cells remained silent: both rhythmic burst and tonic firing responses were affected (Fig. 8C). Reduction of the GRP response by SB 366791 was partially or fully reversible in five cells (Fig. 8A). These results suggest that activation of a TRPV1-like conductance contributes significantly to GRP-induced increases in excitability in most PVT neurons, with no apparent preferential role in rhythmic burst or tonic responding cells.
Fibres displaying GRP-like immunoreactivity innervate select nuclei within the midline and intralaminar thalamus. Perforated patch clamp recording in acute in vitro brain slices showed that exposure to low nanomolar concentrations of GRP induces membrane depolarization and low frequency rhythmic burst or tonic firing in neurons in midline PVT and intralaminar central medial thalamic nucleus. Subsequent analyses of membrane currents in voltage-clamped PVT neurons revealed an underlying postsynaptic BB2 receptor-mediated suppression of a Ba2+-sensitive Kir conductance and simultaneous activation of a non-selective cation conductance with TRPV1-like biophysical and pharmacological properties. A significant reduction in membrane depolarization and rhythmic burst or tonic firing in the presence of a selective antagonist indicated that activation of a TRPV1-like conductance contributes significantly to GRP-induced increases in PVT neuronal excitability.
Our histological data confirm and extend previous observations on the presence of a GRP-like immunoreactive innervation of midline and intralaminar thalamus. It has been suggested that this innervation has its origin in the suprachiasmatic nucleus (SCN), known to contain a subpopulation of neurons that synthesize GRP and to send efferents to select midline and intralaminar thalamic nuclei (Watts et al. 1987; Zoeller et al. 1989; Mikkelsen et al. 1991; Schwartz et al. 2011). However, anatomical studies using anterograde tracers have indicated that the SCN innervation is mainly directed to PVT, and does not target many of the other midline and intralaminar nuclei that also display GRP-like immunoreactive fibres (Watts et al. 1987; Schwartz et al. 2011). Moreover, a combined retrograde tracing and labelling study detected GRP-like immunoreactivity in only a small percentage of PVT-projecting SCN neurons (Novak et al. 2000). The generally restricted thalamic projections of SCN and GRP-immunoreactive SCN neurons therefore suggest the existence of additional sources of GRP in midline and intralaminar thalamus; these may include the parabrachial nucleus and nucleus of the solitary tract (Wada et al. 1990; Krout et al. 2002).
Voltage clamp analyses of membrane currents revealed three aspects of the membrane mechanism underlying GRP's excitatory action on PVT neurons:
- 1A specific involvement of postsynaptic BB2 receptors. This is consistent with the nanomolar affinity of BB2 receptors for GRP (Jensen et al. 2008), yet surprising in view of the absence of BB2 receptors in midline and intralaminar thalamus reported in original studies (Ladenheim et al. 1992; Wada et al. 1992). However, more recent data obtained in mouse brain do suggest the expression of BB2 receptors in this part of the thalamus (http://www.alleninstitute.org). Despite their prominent presence, the absence of an implication of BB1 and BB3 receptors was not unexpected, as they require binding of other endogenous ligands (Ladenheim et al. 1992; Wada et al. 1992; Jennings et al. 2003; Jensen et al. 2008). Yet, it is possible that BB1 receptors may become functionally relevant when GRP concentrations reach the micromolar range (Jensen et al. 2008).
- 2Suppression of a Kir conductance. Evidence that suggests it concerns a Kir2-type conductance includes suppression of GRP's action by Ba2+ in concentrations (30 μm) that preferentially block Kir2 channels, and the lack thereof by agents that selectively block or antagonize Kir3 or Kir6 channels (Hibino et al. 2010). Moreover, Kir2 subfamily subunits are expressed in PVT (Horio et al. 1996). The results are consistent with previous reports on suppression of Kir2 conductances by activation of other Gq/G11 protein-coupled receptors (e.g. Shen et al. 2007).
- 3Activation of a non-selective cation conductance with TRPV1-like properties. This proposal for the conductance's identity is based on both biophysical and pharmacological features that include a characteristic I–V relationship, a selective permeability to Ca2+, inhibition by the selective TRPV1 antagonists CPZ and SB 366791, and occlusion by the prototypical TRPV1 agonist capsaicin. Moreover, TRPV1 expression has been reported, and disputed, in midline and intralaminar thalamus (Mezey et al. 2000; Roberts et al. 2004; Cavanaugh et al. 2011). However, the use of the term ‘TRPV1-like’ is warranted as some properties, including maximum inward currents near VH–40 mV, a pronounced negative slope at VH≤−40 mV and partial inhibition by La3+, are different from previously reported features of TRPV1 currents (Gunthorpe et al. 2000; Clapham, 2003; Wu et al. 2010). One possible explanation for these diverging features could be the existence of a heteromeric ion channel. Thus PVT neurons might express TRPV1 in combination with channel subunits that do show La3+ sensitivity, including members of the same subfamily, such as TRPV2 or TRPV3 (Smith et al. 2002; Liapi & Wood, 2005: Rutter et al. 2005; Clapham, 2007), or members of a different subfamily, such as TRPC1 (Clapham, 2007; Ma et al. 2011). Alternatively, PVT neurons could contain a splice variant of TRPV1 that has different functional properties (cf. Sharif Naeini et al. 2006). A detailed molecular analysis of TRPV and other subfamily members in PVT neurons may assist in distinguishing these possibilities.
The second messenger pathways/molecules implicated in suppression of the Kir conductance and activation of the TRPV1-like conductance remain to be examined. It has been established that BB2 receptors primarily couple to Gq/G11 proteins that activate PLC to generate inositol 1,4,5-triphospate and diacylglycerol from membrane phosphoinositides (Jensen et al. 2008). However, biochemical studies have indicated that BB2 receptor activation may also increase the activity of other phospholipases, including phospholipase A2 producing arachidonic acid and lysophospholipids (Jensen et al. 2008). Such diverging pathways suggest the possibility that BB2 receptor activation might modulate different ionic conductances through in part distinct second messenger molecules. For example, suppression of the Kir conductance in PVT neurons might arise as a consequence of PLC-induced depletion of the membrane phosphoinositide phosphatidylinositol 4,5-biphosphate (Stanfield et al. 2002; Hibino et al. 2010). Activation of the TRPV1-like conductance might result from a similar mechanism, but may also stem from an interaction by lipoxygenase-synthesized hydroperoxyeicosatetraenoic acids from phospholipase A2-produced arachidonic acid (Caterina & Julius, 2001; Chuang et al. 2001; Kauer & Gibson, 2009). The possible involvement of these second messenger pathways may explain why GRP, in contrast to the direct channel interaction by capsaicin, can result in sustained activation of the TRPV1-like conductance.
Both our voltage and current clamp data imply that activation of a postsynaptic TRPV1-like membrane conductance is partly, perhaps primarily, responsible for GRP-induced increases in PVT neuronal excitability. Our results add to recent data on the CNS presence of functional TRPV1 channels in postsynaptic membranes (Sharif Naeini et al. 2006; Chávez et al. 2010; Grueter et al. 2010; Kim et al. 2012), and to evidence for a role of postsynaptic TRPV1 in sustained transmitter-induced increases in CNS neuronal excitability (Sharif Naeini et al. 2006). Because of the strategic anatomical position of PVT and its innervation by SCN (Watts et al. 1987; Mikkelsen et al. 1991; Van der Werf et al. 2002), one might speculate that GRP's modulation of a postsynaptic TRPV1-like conductance, as well as of the Kir conductance, in PVT neurons may be involved in (circadian regulation of) arousal and awareness.
M.L.H.J.H. and L.P.R. conceived the study. M.L.H.J.H. designed and performed the electrophysiological experiments. E.M.C. performed the immunohistochemistry. M.L.H.J.H. and M.K. analysed the data. M.L.H.J.H., M.K., E.M.C. and L.P.R. interpreted the data and drafted the manuscript. All authors approved the final version for publication.
The research was supported by the Canadian Institutes of Health Research (grant no. 77745). L.P.R. holds the J. David Grimes/GlaxoSmithKline/Canadian Institutes of Health Research Chair at the University of Ottawa.