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Proc Natl Acad Sci U S A. Nov 29, 2011; 108(48): 19413–19418.
Published online Nov 15, 2011. doi:  10.1073/pnas.1117020108
PMCID: PMC3228463
Physiology

Hypermorphic mutation of the voltage-gated sodium channel encoding gene Scn10a causes a dramatic stimulus-dependent neurobehavioral phenotype

Abstract

The voltage-gated sodium channel Nav1.8 is known to function in the transmission of pain signals induced by cold, heat, and mechanical stimuli. Sequence variants of human Nav1.8 have been linked to altered cardiac conduction. We identified an allele of Scn10a encoding the α-subunit of Nav1.8 among mice homozygous for N-ethyl-N-nitrosourea-induced mutations. The allele creates a dominant neurobehavioral phenotype termed Possum, characterized by transient whole-body tonic immobility induced by pinching the skin at the back of the neck (“scruffing”). The Possum mutation enhanced Nav1.8 sodium currents and neuronal excitability and heightened sensitivity of mutants to cold stimuli. Striking electroencephalographic changes were observed concomitant with the scruffing-induced behavioral change. In addition, electrocardiography demonstrated that Possum mice exhibited marked sinus bradycardia and R-R variability upon scruffing, abrogated by infusion of atropine. However, atropine failed to prevent or mitigate the tonic immobility response. Hyperactive sodium conduction via Nav1.8 thus leads to a complex neurobehavioral phenotype, which resembles catatonia in schizophrenic humans and tonic immobility in other mammals upon application of a discrete stimulus; no other form of mechanosensory stimulus could induce the immobility phenotype. Our data confirm the involvement of Nav1.8 in transducing pain initiated by cold and additionally implicate Nav1.8 in previously unknown functions in the central nervous system and heart.

Keywords: cold sensitivity, EEG, scruffing

Voltage-gated sodium channels (VGSCs) mediate the rapid influx of sodium ions that underlies the rising phase of action potentials in neurons, muscles, and the heart (1). Each of the nine mammalian VGSCs consists of a pore-forming α-subunit and one or more auxiliary β-subunits (2). The α-subunits contain four homologous domains, each consisting of six transmembrane α-helices that control channel opening, ion selectivity, and inactivation. The β-subunits function to modify the kinetics and voltage dependence of channel gating, channel localization, and interactions with other proteins.

Multiple inherited channelopathies are caused by mutations that render VGSCs either hypo- or hyperexcitable. These channelopathies include epilepsy and Dravet syndrome caused by mutations in Nav1.1 and Nav1.2; hyperkalemic or hypokalemic periodic paralysis syndrome caused by mutations in the skeletal muscle channel Nav1.4; long QT syndrome, Brugada syndrome, and Sudden Infant Death syndrome caused by mutations in the cardiac channel Nav1.5; erythromelalgia and paroxysmal extreme pain disorder caused by mutations in Nav1.7; and altered pain sensitivity caused by mutations in Nav1.8 and Nav1.9 (3). These diseases underscore the importance of VGSCs in physiology, but the full spectrum of events requiring sodium channel function is not definitively known.

Here, we report an allele of Scn10a, termed Possum, that reveals previously unknown roles for Nav1.8. Nav1.8 is an α-subunit of slowly inactivating, tetrodotoxin (TTX)-resistant VGSCs that are most highly expressed in small- and medium-diameter sensory neurons of the trigeminal and dorsal root ganglia (DRG) (4, 5). A null mutation of Scn10a in mice caused reduced responsiveness to thermal and mechanical pain stimuli and abrogated responsiveness to noxious cold and mechanical stimulation at low temperatures (69). Nav1.8 mRNA is also detected in the heart, and several common single-nucleotide polymorphisms have been associated with altered cardiac conduction in humans (1012).

The visible aspects of the Possum phenotype resemble tonic immobility, a behavior likened to the temporary, whole-body arrest of movement that occurs when prey animals come under attack by a predator, which can be produced experimentally in a wide range of species by certain drug treatments, central nervous system (CNS) lesions, or manual restraint procedures (13, 14). Sodium conduction by Nav1.8 may contribute to this poorly understood behavior (SI Discussion).

Results

Possum Mutants Display Inducible Immobility.

The semidominant Possum phenotype was observed within the third generation (G3) of C57BL/6J mice carrying mutations induced by N-ethyl-N-nitrosourea. Both heterozygous and homozygous Possum mice arrest all movement, exhibit transient apnea (5–10 s) and assume a rigid posture following pinching of the skin at the back of the neck (“scruffing”), a common technique used to immobilize mice for various procedures (Movie S1). Scruffed Possum mice were unable to right themselves when placed on their side or back and also displayed “waxy flexibility” in which the tail could retain a manually set position (Fig. 1 AC and Movie S1). Episodes of such immobility were typically 1–5 min in duration (longer in homozygotes, shorter in heterozygotes), after which mice abruptly returned to normal activity with no apparent residual effects. Recovery from immobility could be promoted by auditory (e.g., clapping) or mechanical stimulation (e.g., gentle prodding), and after as little as 30 s, scruffing resulted in a repetition of the behavior. The behavior recapitulates precisely the features of experimentally induced tonic immobility (13, 14). It occurred after the first instance of scruffing even before weaning age, 1 wk being the earliest age at which it was observed (before this time abnormal immobility is difficult to distinguish from normal behavior), and repeated with similar duration in response to scruffing throughout the life of the mouse (to date, up to 1 y of age). Habituation did not occur when mice were scruffed on a daily basis. Scruffing never caused immobility in wild-type mice, but the immobility phenotype was 100% penetrant in mutants. Possum homozygotes displayed normal levels of activity in tests of locomotion (Fig. S1).

Fig. 1.
The Possum phenotype and identification of a mutation in Scn10a. (A) A wild-type mouse and (B and C) a Possum homozygote after scruffing. (B) The Possum homozygote is immobile and displays waxy flexibility (tail holds a raised, kinked position applied ...

Mutation of Scn10a Causes the Possum Phenotype.

The semidominant Possum mutation was mapped to chromosome 9 (Fig. 1D and SI Results). We sequenced all exons and exon boundaries within the 4.67-Mb critical region and identified a single mutation, an A-to-G transition at position 2403 of the Scn10a transcript (Fig. 1E), corresponding to a threonine-to-alanine change at residue 790 of the 1,957-amino-acid Nav1.8 protein. Like other sodium channel α-subunits, Nav1.8 contains four homologous domains (I–IV), each consisting of six transmembrane α-helices (S1–S6). The Possum missense mutation is located in helix S5 of domain II at the cytoplasmic interface of the channel (Fig. 1F). The phenotype was still observed when the Possum allele was placed in trans with a knockout allele of Scn10a, consistent with a strong, dominant hypermorphic effect of the mutation.

Nav1.8Possum-Mediated Currents Are Strongly Up-regulated.

To understand how the Possum mutation affects the function of Nav1.8, whole-cell voltage and current clamp methods were used to characterize Nav1.8 currents and the excitability of acutely cultured homozygous Possum and wild-type small DRG sensory neurons. The Nav1.8 current was isolated from those of other channels, including the other TTX-resistant channel Nav1.9, and the voltage dependence and channel kinetics were determined for the parameters described in Table S1 (Materials and Methods). In wild-type DRG neurons, the peak current density of TTX-resistant Nav1.8-like currents determined using a voltage step protocol was −86 ± 14 pA/pF (mean ± SEM; n = 28) (Fig. 2 A and D; Table S1). Inward currents peaked at −19 ± 2 mV (n = 23) before declining and reversing sign as the driving force on sodium reversed (Fig. 2A, Inset; Vrev, Table S1). Normalizing for driving force on sodium in these experiments, the voltage to half-activate Nav1.8 (V0.5,act) was −29 ± 2 mV (n = 18; Fig. 2E, black circles; Table S1). Similar peak current density was elicited by 1.5 mV/ms voltage ramps from −81.5 mV (−50 ± 14 pA/pF; n = 10). The time to peak, rate of current decay during step depolarizations, and deactivation of peak currents by hyperpolarization were voltage dependent (Table S1). The voltage to half-inactivate peak current after a 30-ms prepulse (Fig. 2C, voltage protocol shown in Inset) was −42 ± 3 mV (V0.5, inact, Table S1; Fig. 2F) with a slope of 6 mV/e-fold (k inactivation, Table S1; Fig. 2C, Inset, and F). The rate of recovery from fast inactivation (τfast, recov) using a two-pulse voltage protocol was 2.0 ± 0.2 ms, and ~90% of the current recovered quickly for test pulses ≤30 ms in duration (Table S1). With longer excursions to peak voltages, a proportion of channels slowly inactivate, and the proportion is dependent on the test potential and duration and the interpulse voltage (Table S1). The values of these parameters are similar to those previously reported (15, 16).

Fig. 2.
Nav1.8Possum current is enhanced compared with wild type. (A–H) Families of whole-cell currents in representative wild-type (A) and Scn10aPsm/Psm (B) small sensory neurons in culture. Currents were elicited by step depolarizations from −91.5 ...

TTX-resistant Nav1.8-like currents expressed in homozygous Possum DRG neurons were fourfold larger than those of wild-type DRG (Fig. 2 B and D; Table S1). A 3.6-fold increase in ramp-induced inward current was also observed (−180 ± 45 pA/pF (n = 6); P = 0.034). During long-duration steps to subpeak voltages, inactivation of Nav1.8-like currents in Possum neurons was impaired compared with that in wild-type neurons (Fig. 2G) due to a 1.6-fold slowing of both fast and slow components of fast inactivation (Table S1) and a twofold increase in the proportion of the slow component (Fig. 2H). There was a significant slowing of activation as well (time to peak, Table S1). No differences between Possum and wild-type DRG neurons were observed for V0.5,act, V0.5,inact, k values, deactivation rates, or recovery from fast or slow inactivation (Fig. 2 E and F; Table S1). Therefore, the Possum mutation increases the current mediated by Nav1.8 channels by increasing the apparent current density and proportion of slow:fast components and by slowing the kinetics of fast inactivation. Because these changes likely heighten the excitability of Nav1.8-expressing neurons, we performed current clamp recordings in the presence of TTX to determine whether Possum neurons are hyperexcitable. Although no detectable differences in resting potential, action potential duration, and membrane resistance were measured at −60 mV, the amount of depolarizing current required to elicit an action potential (rheobase) was profoundly decreased in Possum compared with wild-type neurons as was the voltage threshold and the number of action potentials elicited during 150 and 900 ms of injection of current at twice the rheobase (Table S2). Thus, Possum small DRG neurons with long-duration action potentials are hyperexcitable compared with those of littermate controls.

Nav1.8Possum Mutants Are Hypersensitive to Cold Pain but Not to Other Types of Pain.

Nav1.8 is expressed in DRG (4, 5, 17, 18) where it functions to propagate nociceptive signaling in response to noxious cold, heat, and mechanical stimuli (79). We hypothesized that Possum mice are more sensitive to painful stimuli and that exaggerated nociceptive responses provoked by scruffing or other painful stimuli may trigger immobility in Possum mice in the way that excessive pain may provoke paralysis or fainting in humans. Therefore, we examined pain responses of Possum mice. Consistent with electrophysiological studies of the Nav1.8Possum channel, homozygous Possum mice displayed significantly increased sensitivity in the cold-plate test (Fig. 3A). However, homozygous Possum and wild-type mice exhibited similar latencies to paw withdrawal in the hot-plate test, demonstrating normal pain responses to heat stimuli (Fig. 3B). Paw withdrawal responses elicited by von Frey filaments applied to the plantar surface did not differ between Possum and wild-type mice, either before or after injection of complete Freund's adjuvant into the paw (Fig. 3C). These findings support the conclusion that heightened Nav1.8 conductance specifically sensitizes the perception of pain induced by cold, but not by heat or punctate mechanical stimuli.

Fig. 3.
Heightened sensitivity to cold pain in Possum mice. (A) Number of hind-paw lifting or jumping responses of mice placed on a −1 °C metal plate for 2 min. n ≥ 6/genotype, including male and female littermates. (B) Time interval between ...

None of the above-tested noxious stimuli induced immobility in Possum mice. In addition, treatment of neonatal Possum mice with capsaicin to ablate TRPV1-expressing nociceptive C fibers did not abrogate scruffing-induced immobility [although their responses in the cold-plate test were reduced relative to untreated Possum mice (Fig. S2)], indicating that the phenotype is not a response to pain sensed by capsaicin-susceptible sensory neurons. Neither sudden immersion in cold water nor pinching of the tail or feet induced immobility in Possum mice.

Normal Fear Responses in Nav1.8Possum Homozygotes.

Fear lengthens the duration of manipulation-induced tonic immobility (14), and fear induced by predators has been suggested to be a natural cause of tonic immobility. This raises the possibility that scruffing-induced immobility in Possum mice results from a heightened fear response. However, in tests of conditioned fear elicited by foot shock paired with visual and auditory cues, Possum homozygotes displayed similar or reduced fear responses relative to wild-type mice (SI Results and Fig. S3). Foot shock alone did not induce the immobility phenotype.

Altered CNS Activity During Tonic Immobility of Possum Mutants.

Because our behavioral data did not support the hypothesis that immobility and/or loss of consciousness occurred as a result of excessive pain, we re-examined the Possum phenotype in an effort to understand its mechanism. Electroencephalogram (EEG) activity was measured in homozygous Possum mice before and after scruffing and compared with that of wild-type mice given the same treatment (Fig. 4 and Fig. S4). During the baseline period before scruffing, frontal and parietal EEG patterns were similar in Possum and wild-type mice, with no epileptiform or other abnormal activity. Wild-type mice maintained an identical EEG pattern after scruffing. In contrast, scruffing of Possum homozygotes produced a shift in EEG activity from frequencies predominantly in the 4- to 10-Hz range (θ-rhythm) during baseline recording to δ-frequencies in the 1- to 4-Hz range concomitant with immobility. Coincident with recovery from immobility, EEG activity abruptly returned to and maintained baseline patterns indefinitely. Following a 5-min recovery period, scruffing again resulted in immobility and development of altered EEG activity similar to that observed during the first episode. These data implicate Nav1.8 in modulating the activity of CNS neurons. Interestingly, measurements of evoked response potentials (ERPs) to auditory stimuli in Possum mice suggested that, although immobile, the mice are alert rather than unconscious (Fig. S5).

Fig. 4.
Shift in EEG from θ-rhythm to δ-rhythm upon initiation of tonic immobility in Possum mice. Average parietal–cortical EEG power recorded from homozygous Possum (n = 8) or wild-type mice (n = 8) during the indicated stages. Stages ...

Sinus Bradycardia During Immobility of Nav1.8Possum Homozygotes.

Nav1.8 transcripts have been detected in the heart (10), and three studies reported association between common sequence variants of human SCN10A and prolongation of the PR interval, P wave, and QRS complex on an electrocardiogram (ECG) (1012). It is believed that the channel can act within cardiomyocytes to directly affect their function, possibly by modulating sodium currents or regulating interactions with accessory proteins. We considered that a defect in cardiac conduction might cause or contribute to the Possum phenotype and performed ECG recordings from wild-type and homozygous Possum mice before and after scruffing. Before scruffing, Possum homozygotes exhibited heart rates similar to those of wild-type mice (Fig. 5A). In contrast, the heart rates of Possum mice were reduced by ~50% during the immobile period after scruffing relative to baseline heart rates (Fig. 5A). The ECGs recorded for wild-type and homozygous Possum mice showed no significant differences in PR interval, P wave duration, or QRS duration before scruffing. However, scruffing of Possum mice resulted in irregular RR intervals following clearly defined P waves (Fig. 5B and Fig. S6), consistent with sinus bradycardia with increased heart rate variability coincident with the Possum response. No significant ECG changes were observed following scruffing of wild-type mice. The changes in heart rate and rhythm resolved as the mice became ambulatory. Because bradycardia can cause hypotension and loss of consciousness, we measured the blood pressure of Possum mice but found no fluctuation in blood pressure following scruffing, indicating that immobility is not associated with or caused by changes in blood pressure (Fig. 5C).

Fig. 5.
Sinus bradycardia and irregular RR intervals during immobility of Possum mice. (A) Mean heart rate before and after scruffing of homozygous Possum mice left untreated or injected i.p. with atropine. Each point represents an individual mouse. (B) Representative ...

Because increased vagus nerve discharge can decrease heart rate and cause heart rate variability, we tested whether the bradycardia and marked RR variability in Possum mice resulted from increased vagal tone. Possum homozygotes were treated before scruffing with atropine, an anticholinergic drug that inhibits the action of the vagus nerve on the heart. Atropine prevented the reduction in heart rate (Fig. 5A) and normalized cardiac rhythm during immobility (Fig. 5B and Fig. S6), but failed to prevent the Possum immobility response itself. No ECG changes were observed with scruffing or atropine administration in wild-type mice. Thus, whereas excessive vagal output to the heart is responsible for bradycardia and RR variability in Possum mice, it is not responsible for the tonic immobility induced by scruffing. Neither epinephrine nor propranolol, administered at large pharmacologic doses, abolished Possum behavior or induced it in wild-type mice.

Discussion

The Possum mutation, a threonine-to-alanine substitution in transmembrane helix S5 in domain II, alters the physiology of Nav1.8 channels by increasing current density and by slowing the kinetics of inactivation. Precedent exists for the hyperactivation of sodium channels by dominant missense mutations located adjacent to the cytoplasmic leaflet of the plasma membrane lipid bilayer. For example, T704M and M1592V, the two most common mutations of Nav1.4 causing dominant hyperkalemic periodic paralysis syndrome (HYPP), are each located near the intracellular surface of the plasma membrane (19). Similar dominant mutations in Nav1.7 cause erythromelalgia (20, 21). These mutations increase neuronal excitability by shifting the voltage dependence of activation in the hyperpolarizing direction and/or by impairing inactivation. Together, these findings support the idea that residues in predicted S5 regions close to the plasma membrane–cytoplasm interface are critical determinants of channel activation and inactivation properties. Nav1.8 T790 is conserved in all nine mammalian VGSCs in both humans and mice, as are 7 of the 10 surrounding amino acids. Notably, T790 is orthologous to T704 of Nav1.4 on sequence alignment (as noted above, it is one of the mutations causing HYPP). It is likely that alanine, which is nonpolar and hydrophobic, fails to form the electrostatic interactions normally mediated by the polar threonine residue, thereby changing channel conformation and properties. Another factor that may be involved in the effect of the Possum mutation is the change in the size of the amino acid side chain. Side-chain size of S241 in the domain I S4–S5 linker of Nav1.7 was found to be a critical determinant of channel properties (22).

Possum homozygotes display hypersensitivity to cold, consistent with the insensitivity to cold displayed by Nav1.8-null mice (8), firmly establishing Nav1.8 as necessary and sufficient for transduction of noxious cold stimuli. Equally interesting is that we did not observe generally heightened pain sensitivity, for example, to heat or mechanical force. This may indicate a specific role for Nav1.8 in cold transduction. Alternatively, other ion channels/mechanisms may compensate for the hyperconductivity of Nav1.8 during heat and mechanical transduction, although not during transduction at cold temperatures. Future experiments could test whether in vivo electrophysiological recordings manifest the specific cold sensitivity that we observe behaviorally. Importantly, Possum mice may serve as a useful model for the study of cold sensitivity (wild-type mice are notoriously unresponsive to noxious cold stimuli compared with noxious heat in behavioral studies).

The normal responses of Possum mice to noxious heat and punctate mechanical stimuli argue against the idea that excessive pain exists and that it triggers the immobility phenotype in scruffed Possum mice. Among DRG, Nav1.8 is predominantly expressed in nociceptive neurons with C fibers and Aδ-fibers and less in neurons with Aα/β-fibers (5). We showed that nociceptive C fibers ablated by capsaicin are not required for the induction of immobility in Possum mice. We therefore hypothesize that sensory fibers resistant to neonatal capsaicin ablation, which include Aδ-fibers and Mrgprd+ mechanosensitive C fibers (23), play an integral role in the immobility response to scruffing and may be necessary and sufficient for sensing and propagating the signals induced by such physical manipulation. However, it cannot be ruled out that developmental compensatory mechanisms impart susceptibility to the immobility phenotype or that the phenotype is mediated by events other than acute activation of mutant Nav1.8 channels in nociceptors during scruffing.

It was reported previously that nonsynonymous coding sequence variants of human SCN10A are associated with increased PR interval, P wave duration, and QRS duration in ECGs and that Nav1.8-deficient mice have decreased PR intervals (1012). We found that scruffed Possum mice experienced vagus nerve-stimulated sinus bradycardia and highly irregular RR intervals on ECG concomitant with immobility. Our data do not permit us to discern whether the effects of Nav1.8Possum on cardiac conduction are intrinsic to cardiomyocytes or secondary to neurotransmission via DRG neurons. However, observation of normal blood pressures during immobility and the inability to block immobility with atropine support the conclusion that the cardiac abnormalities are independent of the behavioral Possum phenotype. The ability to restore normal cardiac rhythm and prevent bradycardia using atropine demonstrates the existence of a unique reflex arc activated by a discrete stimulus (scruffing) and having as one output a strong vagal response.

We showed that neither excessive pain nor altered cardiac conduction was responsible for the immobility phenotype, yet the mechanistic basis of the behavior remains unknown. We hypothesize that scruffing-induced immobility in Possum mice is equivalent to the phenomenon of manipulation-induced tonic immobility and, as such, may be mediated by excessive neuronal transmission in the parabrachial region of the brainstem and the ventrolateral periaqueductal gray of the midbrain upon activation by Nav1.8Possum-expressing DRG neurons (SI Discussion). Altered EEG patterns, observed only during immobility, also raise the possibility of direct function(s) for Nav1.8 in CNS neurons, which may contribute mechanistically to the behavior. Although brain expression of Nav1.8 has not been reported (4, 5, 17, 18), small amounts or highly localized expression below the level of detection by standard assays cannot be ruled out.

We note that the immobile state induced by scruffing of Possum mice shares striking similarity with aspects of the human syndrome of catatonia, which include the symptoms of immobility, mutism, and waxy flexibility and are observed in patients with schizophrenia, schizoaffective disorder, and other neurological illnesses (24).

Materials and Methods

Animals.

Mice were maintained at The Scripps Research Institute under standard housing conditions, and all procedures were approved and performed according to institutional guidelines for animal care. Scn10atm2(cre)Jnw mice (MGI:3053096) were a gift from John Wood (University College London). C57BL/6J mice were mutagenized with N-ethyl-N-nitrosourea as previously described (25) and Possum variants were observed among G3 mice of a single pedigree and expanded to form a stock on the basis of the visible behavioral phenotype. The Possum strain (Mutant Mouse Regional Resource Center #030627) is described at http://mutagenetix.utsouthwestern.edu/home.cfm. Mapping and DNA sequencing were performed as described (http://mutagenetix.utsouthwestern.edu/protocol/protocol_rec.cfm?pid=14).

Electroencephalography.

Surgical procedures and electrophysiological (EEG and ERP) recordings were performed as described (26). A three-tone auditory “oddball” paradigm that has been developed to directly model studies used in humans was employed (27). The latency windows were as follows: P1, 25–65 ms; N1a, 10–50 ms; N1b, 50–150 ms; P2, 100–250 ms; N2, 200–300 ms; and P3, 265–355 ms. Further details about auditory ERP sessions and electrophysiological analyses were described previously (26).

Electrophysiology.

We recorded Nav1.8-mediated currents from acute cultures of neurons extirpated from cervical, thoracic, and/or lumbar DRG of adult mice and prepared them as described (28) using published methods (16, 2931) (SI Materials and Methods). Glial cell-derived neurotrophic factor was omitted from the culture media to maintain low Nav1.9 expression (32). Nav1.8-mediated currents in neurons from DRG innervating the skin of the neck [C2-5 and T1 (33)] and T2-L5 were similar, and data were combined. Neurons from Possum mice were compared with C57BL/6 controls or wild-type littermates with equivalent results. The whole-cell configuration of the patch clamp technique was performed using a Multiclamp700A amplifier and Digidata 1322A as described (34) on small- to medium-diameter (19–35 μm) DRG neurons 8–48 h after plating with modifications for recording voltage-gated TTX-R currents as described in SI Materials and Methods.

Electrocardiography and Blood Pressure Measurement.

For surface ECG recording, 30-gauge needle limb electrodes were inserted s.c. under anesthesia (1.5% isoflurane). Following electrode placement, isoflurane administration was withheld, and ECG was recorded while mice were awake at baseline and during immobility. ECG signal was amplified using a Warner Instruments DP-304 differential amplifier band pass filtered between 0.1 and 100 Hz. Signal was further filtered through a Quest Scientific HumBug 50/60 Hz Noise Eliminator and digitized at 3,000 Hz. Data were acquired and analyzed using QRS Analysis Software (QRS Phenotyping) (35). Possum mice were compared with outbred controls.

Telemetry transmitters TA10ETA-F20 (DSI) were implanted for continuous blood pressure and heart-rate monitoring. Surgical insertion of the arterial catheter with transducer and data acquisition were performed as described (36). Blood pressure and heart rate were recorded at baseline and during immobility before and after i.p. atropine (1 mg/kg) administration.

Analysis of Response to Painful Stimuli and Conditioned Fear.

Mice were first habituated in testing chambers. For thermal pain, mice were placed on a 52 °C hot plate (Ugo Basile). The time of onset was recorded as the first response, such as a paw flick, paw lick, paw lift, or jump. To evaluate cold pain, mice were placed onto a −1 °C cold plate (TECA) for 2 min. The number of responses of paw flicks, paw licks, paw lifts, or jumps were recorded. For mechanical pain, a Von Frey apparatus (Dynamic Plantar Aesthesiometer, UGO Basile) was used as described (37). In some experiments, 2-day-old Possum mice were injected s.c. with 50 mg/kg capsaicin and tested at least 8 wk later for responses to cold stimuli or scruffing. Conditioned fear testing was performed in Freeze Monitor chambers (San Diego Instruments) housed in sound-proofed boxes as described (38).

Statistical Analysis.

Statistical analyses of EEG and ERP recordings were performed by using SPSS for the Macintosh (SPSS, Inc.). Brain regions were assessed independently. Groups (B6 vs. Possum mice) were assessed as a between-subject variable. Tone (standard, rare, and noise) was assessed as within-subject repeated measures. For in vitro electrophysiological properties of DRG, the statistical significance of differences was determined by the two-tailed Student's t test. For all other data, the statistical significance of differences was determined by one-way ANOVA. P value was set at P < 0.05 to determine the levels of statistical significance.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Dr. Bertrand Coste for helpful discussion; Taryn Earley, Yusu Gu, Corinna Kimball, and Chay Bae for excellent technical assistance; and Diantha La Vine for assistance with figure preparation. This work was supported by National Institutes of Health Grant AA006059 (to C.E.) and Broad Agency Announcement Contract HHSN27220000038C (to B.B.). A.L.B. was supported by The Irvington Institute Fellowship Program of the Cancer Research Institute.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1117020108/-/DCSupplemental.

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