Lower core body temperature and attenuated nicotine-induced hypothermic response in mice lacking the β4 neuronal nicotinic acetylcholine receptor subunit

Brain Research Bulletin 66 (2005) 30–36

Lower core body temperature and attenuated nicotine-induced hypothermic response in mice lacking the ␤4 neuronal nicotinic acetylcholine receptor subunit Ram Sack a,b,1 , Alona Gochberg-Sarver a,b,1 , Uri Rozovsky a , Merav Kedmi a,b , Serena Rosner a , Avi Orr-Urtreger a,b,∗ a

The Genetics Institute, Tel-Aviv Sourasky Medical Center, 6 Weizmann St., Tel Aviv 64239, Israel b Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel

Received 8 December 2004; received in revised form 16 February 2005; accepted 27 February 2005 Available online 22 April 2005

Abstract Diverse physiological and pathological effects of nicotine, including the alteration of body temperature, are presumably mediated by neuronal nicotinic acetylcholine receptors (nAChR). Previous studies have suggested the involvement of distinct nAChR subunits in nicotineinduced thermoregulation. We studied genetically manipulated knockout mice lacking the ␣7, ␣5 or ␤4 subunit genes, in order to assess the effects of subunit deficiency on temperature regulation. Using a telemetry system, core body temperature was monitored continuously prior to and following nicotine administration in mutant mice and in wild-type littermates. Mice lacking in the ␤4 nAChR subunit gene had significantly lower baseline core body temperature than all other mouse strains studied. ␤4 null mice also demonstrated a reduced nicotineinduced hypothermic response and impaired desensitization following repeat nicotine exposure. These findings suggest the involvement of the ␤4 nAChR subunit in both core body temperature homeostasis and nicotine-elicited thermo-alterations in mice. © 2005 Elsevier Inc. All rights reserved. Keywords: Nicotinic acetylcholine receptors; ␤4 subunit; Temperature regulation; Nicotine-induced hypothermia; Knockout mice

1. Introduction The neuronal nicotinic acetylcholine receptors (nAChRs) belong to the large super family of ligand-gated ion channels and are expressed throughout both the central and peripheral nervous systems, and in non-neuronal cells (reviewed by [2]). To date, 12 distinct genes encoding neuronal nAChR subunits have been identified, 9 ␣-type (␣2–␣10) and 3 ␤type (␤2–␤4), generating an abundance of structurally and functionally distinct hetero- and homo-pentameric receptors (reviewed by [9]). The neuronal nAChRs mediate the effects Abbreviations: nAChR, neuronal nicotinic acetylcholine receptors; Tc , core body temperature; WT, wild-type ∗ Corresponding author. Tel.: +972 3 697 4704; fax: +972 3 697 4555. E-mail address: [email protected] (A. Orr-Urtreger). 1 Authors contributed equally to this manuscript. 0361-9230/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2005.02.032

of the endogenous neurotransmitter acetylcholine, and are the principal biological targets of the tobacco alkaloid, nicotine. Nicotine has diverse effects on the nervous system, altering behavior and cognition [16], inducing seizures [17], mediating analgesia [26] and regulating autonomic functions [6,41]. Nicotine also elicits changes in core body temperature. Early experiments demonstrated the induction of a hypothermic response following intracerebral nicotine administration in cats [10], monkeys [11,12] and rats [15]. The significant tolerance to the hypothermic effects of an acute nicotine challenge following chronic systemic nicotine infusion in mice [5,19,34] as well as reported time and dose dependencies, support a receptor mediated process [5,23,24]. While the molecular mechanisms underlying mammalian thermoregulation are largely unknown, data suggest that nicotineinduced alterations in body temperature are influenced by genetic factors. Differential sensitivity to dose dependent

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nicotine-induced hypothermia and acute tolerance has been identified in different strains of inbred mice [3,20,22,25]. Furthermore, the missense mutation Charna4 A529T identified in the murine ␣4 nAChR subunit gene was associated with increased sensitivity to various effects of nicotine, including nicotine-induced hypothermia [35,37,39]. A recent study in knockout mice deficient in the ␤2 nAChR subunit reported a reduced hypothermic response to low doses of nicotine, suggesting that this subunit partially mediates nicotine-induced hypothermia [38]. The objective of the present study was to evaluate the possible involvement of various neuronal nAChR subunit genes in temperature homeostasis and in nicotine-induced hypothermia using mutant mice deficient for ␣7, ␣5 or ␤4 subunits (␣7−/−, ␣5−/− and ␤4−/−, respectively). Significant alterations in both baseline core body temperature and in the hypothermic response normally induced by nicotine were observed in mice deficient in the ␤4 subunit gene, suggesting that this nAChR subunit plays an important role in mammalian core body temperature homeostasis and in the mediation of nicotine-induced temperature changes.

2. Methods 2.1. Animals All mice used in this study were from congenic lines that were backcrossed onto a C57BL/6J background for at least six generations after germline transmission. Mutant mice were homozygous for a null mutation in the ␣7 (n = 11) [29], ␣5 (n = 10) [32] or ␤4 nAChR subunits (n = 21) [43]. Wildtype littermates from the mutant strains were used as controls (n = 15). Experiments were performed on a total of 57 mice (31 males and 26 females). Average age and weight were 13 ± 4.9 weeks and 24 ± 3.8 g, respectively. Prior to the experiments, 2–5 mice were housed in each cage, under a 12-h light:12-h dark cycle in temperature-regulated room (22–24 ◦ C), with food and water ad libitum. All procedures were approved by the institutional animal and care committee, in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Each mouse was genotyped twice, once before and once following the experiment. The genotyping of ␣5 and ␤4 null mice and WT mice was as described previously [14]. ␣7 null mice were genotyped using three-way PCRs with primers sequences as follows: ␣7 forward, 5 GGTTTCCTGGTCCTGCTGTGTTA-3 ; ␣7 WT reverse, 5 CTGCTGGGAAATCCTAGGCACACTTGCG-3 ; ␣7 mutant reverse, 5 -GGCAGAGGTTACGGCAGTTTGTC-3 . All PCR reactions were performed in a total volume of 25 ␮l, using 1.25 units of Taq DNA polymerase (Sigma–Aldrich, St. Louis, MO, USA) and 500–1000 ng genomic DNA as a template, in a T3 Thermocycler (Biometra® , G¨ottingen, Germany). The PCR conditions for ␣7 genotyping included an initial denaturation step of 95 ◦ C for 2 min followed by 35

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cycles, each of 94 ◦ C for 45 s, 62 ◦ C for 45 s and 72 ◦ C for 45 s, and a final extension step of 72 ◦ C for 5 min. 2.2. Telemetry The telemetry system (DATA Sciences’ PhysioTel® Telemetry, St. Paul, MN, USA) was used to measure physiological parameters including temperature and heart rate. The system includes an implantable transmitter chip (radiofrequency transducer model TA10ETA-F20), a receiver panel (RPC-1), a consolidation matrix and a personal computer with accompanying Dataquest Data Analysis software (Dataquest A.R.TTM , Version 2.10, St. Paul, MN, USA), allowing continuous recording in conscious, unrestrained and uninterrupted stress-free animals. 2.3. Surgical procedures Mice were anaesthetised by an intra-peritoneal (i.p.) injection of Ketamine HCl/Rumpon (FORT DODGE® , Iowa, USA), and the telemetry transmitter chip was secured in the intra-peritoneal cavity. Positive and negative leads were directed subcutaneously to the left groin and right shoulder areas, respectively, and securely sutured. Incisions were closed and the animals were returned to individual cages where they recovered in a warm environment until fully awakened. For the remainder of the experiment, mice were caged individually. 2.4. Temperature recording Immediately following the surgical procedure continuous data sampling was initiated, with 10 s fragments recorded every 10 min. After a designated 3-day recovery period, baseline temperature values were recorded for 3 consecutive days (4th, 5th and 6th post-operative days, a total of 72 h). 2.5. Drug administration A single dose of 1 mg/kg body weight (−)-Nicotine (Sigma–Aldrich® , Rehovot, Israel), dissolved in sterile isotonic saline (B. Braun, Melsungen, Germany), was injected intraperitoneally at volume of 0.01 ml/g body weight once daily on the 7th, 8th and 9th post-operative days, and temperature values were recorded in 10 s fragments each minute for 3 h, 1 h prior to and 2 h following i.p. nicotine injection. Saline administration at the same volume per body weight served as a control. The magnitude of nicotine-induced hypothermic response in each mouse was determined by calculating the area under the curve (AUC) 5–120 min after i.p. nicotine injection. The following formula calculated AUC based on the area generated by the differences between the baseline temperature (before nicotine injection) and the temperature nadir at each minute: AUC = 115a − [(b + c + 2d)/2] (AUC, arbitrary units), where a is the average baseline temperature

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5–60 min prior to nicotine injection, b the temperature value 5 min post-nicotine injection, c the temperature value 120 min post-nicotine injection and d is the sum of temperature values 6–119 min post-nicotine injection. The values of the first 5 min after nicotine injection were not included in the calculation in order to avoid artifactual measurements resulting from handling the mice during the injection. 2.6. Statistical analysis The basic statistical model was analysis of variance (ANOVA). When necessary, we used planed comparison to analyze a specific hypothesis by constructing a contrast, and post hoc analysis to locate a source of significance, using the Tukey algorithm. A p-value of less than 0.05 was considered statistically significant. Pearson correlation was calculated between variables on an interval scale and the Fisher exact test was applied for a-parametric comparison of groups that included less than 25 mice. Statistical analysis was performed using SPSS V12 software (SPSS Inc., Chicago, IL, USA). Graphs were constructed with Prism software (Prism, GraphPad 4 Software, San Diego, CA, USA). Data are expressed as means and standard deviations.

3. Results 3.1. Diurnal/nocturnal core body temperature periodicity is maintained in WT, α7−/−, α5−/− and β4−/− mice Following the designated 3-day recovery period, baseline core body temperatures (Tc ) were recorded continuously for a 72 h period (4th, 5th and 6th post-operative days) in WT (n = 10), ␣7−/− (n = 10), ␣5−/− (n = 9) and ␤4−/− (n = 17) mice (Fig. 1). Since age, weight and gender may influence

Fig. 1. A 72-h recording of core body temperature in wild-type (n = 10), ␣7−/− (n = 10), ␣5−/− (n = 9) and ␤4−/− (n = 17) mice. The diurnal/nocturnal temperature variations, with day Tc lower than night Tc , were maintained in all mice over 3 consecutive days. The average baseline temperature of the ␤4−/− mice was consistently lower than baseline temperatures of all other genotypes tested.

Fig. 2. Average day, night and 24 h core body temperature on the 5th postoperative day. Bar histogram represents mean core body temperature ± S.D. White, black and gray bars represent the average day (07.00–19.00), night (19.00–07.00) and 24 h (7.00–7.00) temperatures, respectively. In all four groups of mouse genotypes, day Tc was significantly lower than night Tc (* p < 0.0001). ␤4−/− mice day, night and average 24 h core body temperature was significantly lower ( p < 0.0001).

body temperature, we examined the possibility of a potential confounding effect of each of these variables. No significant difference was found between the male to female ratio in each group of mice tested (44–60% males, p = 0.874). In a detailed analysis of all data recorded on the 5th post-operative day, no correlation was found between age and weight and the mean Tc (rp = −0.205 and −0.266, respectively, Pearson Correlation test). Interestingly, a significantly lower mean core body temperature was found in all males tested when compared to all females (36.23 ± 0.60 and 36.73 ± 0.46 ◦ C, n = 25 and 21, respectively, p = 0.002) independent of the genotype (WT or mutants, F(3,38) = 1.63, p = 0.19). The gender of mice was therefore entered as a covariate in all statistical analyses and all results are reported after controlling for this variant. The diurnal/nocturnal Tc pattern was maintained in all strains of mice tested during the 72 h recording with day Tc lower than night Tc (Fig. 1). The average day (all data points from 07.00 to 19.00) and night (all data points from 19.00 to 07.00) Tc were evaluated on the 5th post-operative day (Fig. 2). Differences between these average temperatures were analyzed using ANOVA in a mixed inter-intra subject model with two independent variables: time (day versus night) as a repeated (within) subject variable, and strain of mice (WT, ␣7−/−, ␣5−/− or ␤4−/− mice) as an inter (between) subject variable. Day Tc was significantly lower than night Tc (F(1,41) = 43.55, p < 0.0001) in all groups of mice tested (Fig. 2). The difference between day and night Tc in WT mice was more pronounced when compared to the differences observed in ␣7, ␣5 and ␤4 null mice (0.88, 0.70, 0.52 and 0.59 ◦ C, respectively, F(3,41) = 3.235, p = 0.032). 3.2. β4 nAChR subunit null mice have a significantly lower baseline core body temperature The average core body temperature of ␤4−/− mice was lower than the average Tc of WT control mice and of ␣7

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Table 1 Differences in basal core body temperature and nicotine-induced hypothermic response between wild-type mice and mice with nAChR ␣7, ␣5 or ␤4 subunit deficiency Genotype

Wild-type vs. ␣7−/− Wild-type vs. ␣5−/− Wild-type vs. ␤4−/− ␤4−/− vs. ␣7−/− ␤4−/− vs. ␣5−/− ␣7−/−vs. ␣5−/−

Average differences in Tc

Average differences in AUC1

Difference

p-Value

Difference

p-Value

0.35 −0.08 0.82 −0.47 −0.89 −0.42

0.37 0.98 <0.0001 0.075 <0.0001 0.22

20.89 37.93 88.19 −67.30 −50.26 17.04

0.86 0.45 0.009 0.12 0.31 0.93

On the the 5th post-operative day, mean core body temperatures were evaluated for each group of mice, and differences were compared. Significant Tc differences were detected between ␤4−/− and wild-type and ␣5−/− mice, and nearly significant differences were found when comparing ␤4−/− and ␣7−/− mice. There were no differences between the Tc of wild-type, ␣7−/− and ␣5−/− mice (ANOVA followed by Tukey post hoc analysis). The magnitude of nicotine-induced hypothermia was calculated for each group of mice, using the difference between area under the baseline curve (AUC) prior to and following the 1st nicotine injection (see text for detailed explanation). Differences in AUC between the four different groups calculated shows that ␤4−/− mice have a significantly attenuated nicotine-induced hypothermic response compared to wild-type mice (ANOVA followed by Tukey post hoc analysis).

and ␣5 null mice during a 72 h recording (Fig. 1). The average day (07.00–19.00) and night (19.00–07.00) Tc evaluated on the 5th post-operative day revealed significant differences between genotypes (F(3,41) = 10.85, p < 0.0001, Fig. 2). The average 24 h core body temperature of ␤4−/− mice was significantly lower than that of WT control mice and ␣5 null mice and nearly significantly lower than ␣7 deficient mice (p < 0.0001, p < 0.0001 and p = 0.075, respectively; Table 1 and Fig. 2). A significantly lower Tc was also evident in ␤4−/− mice when day and night temperatures were compared independently (F(3,41) = 10.19, p < 0.0001 and F(3,41) = 10.07, p < 0.0001, respectively), and when male and female mice were analyzed separately (F(3,21) = 6.23, p = 0.003 and F(3,17) = 7.78, p = 0.002, respectively).

The Tc response of mice to daily nicotine injections for 3 consecutive days was also examined. Fig. 4A–E demonstrates the Tc responses for each group of mice following both days 1 and 3 nicotine injections as determined by calculating the AUC (area under the curve). Repeated nicotine exposure diminished the hypothermic response in all strains (F(1,39) = 5.23, p = 0.028). Although no interaction was found between the genotype of mice and repeated nicotine injections (1st versus 3rd injection, F(3,39) = 1.33, p = 0.28), the absence of an attenuated response following the 3rd nicotine injection was apparent in ␤4−/− mice relative to WT mice (p = 0.005). Contrast analysis comparing all null mutants to WT controls supported the observation that ␤4−/− mice showed a reduced response to recurrent nicotine injections.

3.3. Mice with β4 nAChR subunit deficiency demonstrate an attenuated nicotine-induced hypothermic response WT (n = 15), ␣7−/− (n = 9), ␣5−/− (n = 10) and ␤4−/− (n = 9) mice received nicotine (1 mg/kg) once a day for 3 consecutive days (7th, 8th, 9th post-operative days). Fig. 3 shows the core body temperature response of the four groups of mice tested following the 1st nicotine injection. In all groups of mutant and control mice tested, there were no statistical differences between the average baseline temperatures (5–20 min prior to nicotine injection) and the average temperatures 105–120 min after nicotine injection (WT: 36.51 ± 0.84 and 36.12 ± 0.48; ␣7: 36.05 ± 0.71 and 35.94 ± 0.55; ␣5: 36.48 ± 0.68 and 36.83 ± 0.82; ␤4: 35.84 ± 0.53 and 35.50 ± 1.24, respectively). One way ANOVA with the AUC as the dependent variable and the genotypes of mice as the independent variable revealed a differential response to nicotine (F(3,42) = 3.88, p = 0.016). Tukey post hoc analysis comparing mutants and WT mice identified the source of significance as the reduced hypothermic response of ␤4−/− mice to nicotine (p = 0.009, Table 1).

Fig. 3. Nicotine-induced hypothermia in wild-type, ␣7−/−, ␣5−/− and ␤4−/− mice. Core body temperatures were measured 1 h prior to and 2 h following nicotine injection. Saline was administrated to ␤4−/− mice as a control. Graph represents the average temperatures of wild-type (n = 15), ␣7−/− (n = 9), ␣5−/− (n = 10) and ␤4−/− (n = 9) mice. Arrow represents the time of nicotine/saline administration.

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Fig. 4. Nicotine-induced hypothermia in wild-type, ␣7−/−, ␣5−/− and ␤4−/− mice following the 1st and 3rd nicotine injections. Graph represents the average temperatures 1 h prior to and 2 h following the 1st (represented by black solid line) and the 3rd (represented by dotted gray line) nicotine injections. (A) Wild-type mice (n = 15), (B) ␣7−/− mice (n = 9), (C) ␣5−/− mice (n = 10) and (D) ␤4−/− mice (n = 9). Arrow represents the time of nicotine administration. (E) Bar histogram represents AUC ± S.D. following the 1st and 3rd nicotine injections in each of the groups of mice tested (1st nicotine injection: wild-type = 249.43 ± 89.34, ␣7 = 228.54 ± 30.85, ␣5 = 211.50 ± 45.32 and ␤4 = 161.24 ± 41.40, and 3rd nicotine injection: wild-type = 178.02 ± 72.58, ␣7 = 176.92 ± 65.94, ␣5 = 185.91 ± 38.53 and ␤4 = 146.46 ± 46.03). The difference in the magnitude of nicotine-induced hypothermia between the 1st and 3rd nicotine injections in ␤4−/− mice was significantly less than that of wild-type mice (* p = 0.005).

4. Discussion The central nervous system and a range of peripheral autonomic mechanisms maintain mammalian core body temperature within a narrow range [1]. Although molecular mechanisms underlying temperature homeostasis are poorly understood, they likely involve multiple genes. A wide variety of pharmacological agents elicit alterations in core body temperature. The hypothermic effects of nicotine have been demonstrated in numerous animal studies

and differential sensitivity to nicotine-induced hypothermia documented in several inbred strains of mice, suggests that a genetic component influences nicotine-induced responses [20,22,25]. Presuming that the effects of nicotine on body temperature are mediated through nAChRs, our objective was to study the association between specific neuronal nAChR subunit genes and temperature regulation, aiming to further clarify the genetic mechanisms underlying this complex process. We studied knockout mice deficient in ␣7, ␣5, or ␤4 subunit genes, comparing these null mutants to

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WT littermates. To the best of our knowledge, our results represent the first evidence of a role for a nAChR subunit gene in baseline homeostasis of core body temperature with ␤4 null mice exhibiting significantly lower core body temperatures. We initiated the study of temperature homeostasis in WT and mutant mice by observing diurnal/nocturnal temperature variability. Rodents maintain low diurnal and higher nocturnal core body temperatures, regulated by the hypothalamic suprachiasmic nuclei (SCN) [27]. The abundant expression of nAChR subunits, particularly ␣7, in the SCN [28], suggests their possible involvement in the maintenance of periodicity of body temperature. We hypothesized a potential disturbance in periodicity in mutant mice, specifically ␣7 null mice, but this was not confirmed. The temperature pattern recorded demonstrated the conservation of low diurnal and higher nocturnal temperatures demonstrated in all groups of mutant mice tested, implying that the deficiency of either the ␣7, ␣5 or ␤4 nAChR subunit gene does not change the diurnal/nocturnal variation of murine body temperature. However, mice deficient for the ␤4 nAChR subunit gene demonstrated diurnal and nocturnal baseline core body temperatures that were significantly lower than those of WT controls, suggesting that nicotine receptors assembled with the ␤4 subunit play a role in the maintenance of core body temperature. Given the established association between body temperature and heart rate [8], we compared heart rate in all groups of mice tested, as a possible explanation for the significantly lower baseline core body temperature in ␤4 null mice. Paradoxically, the ␤4 null mice had significantly higher heart rates (data not shown) when compared to WT controls, and to ␣7 and ␣5 null mutant mice, dismissing bradycardia as the cause of hypothermia in ␤4−/− mice, and suggesting that the increased heart rate might possibly attempt to compensate for the lower core body temperature in ␤4−/− mice. Our study also included the administration of 1 mg/kg body weight of nicotine to WT, ␣7−/−, ␣5−/− and ␤4−/− mice. ␤4 null mice exhibited a significantly attenuated nicotine-induced hypothermic response, when compared to WT mice and to ␣7 and ␣5 null mice, further supporting a role for the ␤4 nAChR subunit in murine thermoregulatory mechanisms. Previous studies suggest that the ␤4 subunit largely influences nAChR affinities and sensitivities to agonists and antagonists [4,18,30,40], and dictates nAChR activation kinetics, conductance and channel open time [13,30,33,36,42]. The absence of the ␤4 subunit likely induces conformational changes in nAChRs, altering receptor function and possibly modifying sensitivity to receptor ligands [40]. In fact, studies in ␤4 null mice have demonstrated decreased sensitivity to both central and peripheral effects of acute nicotine or nicotine agonists, including significantly reduced sensitivity to nicotine-induced seizures [14,31], bladder contractility [7,43] and ileal contractile responses [40]. We suggest that in the current experiment, as in the above reports, the attenuated nicotine-induced hypothermic response in ␤4−/− mice

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is the result of reduced receptor sensitivity to nicotine due to the deficiency of the ␤4 nAChR subunit. Decreased sensitivity to the hypothermic effects of nicotine following repeated administration has been well documented in animal studies [5,21]. We examined nicotineinduced temperature changes in mice injected with nicotine for 3 consecutive days. While repeated nicotine administration ameliorated the nicotine-induced hypothermic response in WT, ␣7−/− and ␣5−/− mice, no significant alteration in nicotine-induced hypothermia was witnessed in ␤4−/− mice, suggesting the possible involvement of this subunit in receptor desensitization. In summary, we have presented the first evidence for the involvement of the ␤4 nAChR subunit in molecular mechanisms underlying core body temperature homeostasis and nicotine-induced hypothermia in rodents, and possibly in humans as well.

Acknowledgements This work was supported by the M.K. Humanitarian Fund. This work was performed in partial fulfillment of the requirements for a Ph.D. degree of Alona Gochberg-Sarver, Sackler Faculty of Medicine, Tel Aviv University, Israel.

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