<i>TailTimer</i> device components.

(TIFF)</p

Experimental timeline.

Baseline latency measures were collected via TailTimer across the first two consecutive days. On the following day, TailTimer was used to measure tail withdrawal latency in the SD males under four different water temperatures (47, 48, 49, and 50°C) while the water mixed at a low, fixed rate. Next, latencies were measured under high, low, and still water mixing speed conditions while the temperature was held constant at ± 0.25°C. On days 4 and 5, tail withdrawal latencies were measured via TailTimer in the SD males at 15 min, 1 h, and 4 h following oral gavage of distilled water or oxycodone (3 mg/kg), respectively. (TIF)</p

Components of the <i>TailTimer</i> device for use in the tail immersion assay.

[A] The Raspberry Pi 3 computer. [B] The TailTimer software in operation. [C] Thermal probe to detect water temperature. [D] Electrical ground wire that remains immersed in the water with the thermal probe (C). [E] Scannable RFID implanted subcutaneously in the rat for identification. [F] Electrical latency wire to be dipped into and withdrawn from the water simultaneously with the rat’s tail to start and stop the timer, respectively. [G] USB RFID scanner. [H] Scannable RFID command fobs used to navigate the TailTimer program in place of a keyboard and mouse. The six necessary fobs are used to enter the user ID, set the target temperature, start a new rat, test the same rat again, delete the last latency, and exit the program.</p

Latency varies across water conditions.

[A] Tail withdrawal latency measured at different water temperatures. Each data point (+) represents one of the 2–3 latency measures obtained for each individual rat at each temperature tested. As water temperature is decreased, latency is lengthened. Linear regression indicated that temperature explains 63% of the variance in latency when tested between 47–50°C with the water mixing at a constant, low speed. [B] Tail withdrawal latency measured at a constant temperature (48°C ± 0.25) and different water mixing speeds. Lengthening of latency occurs as the water mixing speed is decreased with the longest latencies occurring when the water is still (i.e., not being mixed by the stir bar). Data are expressed as mean ± SEM; **p = 0.001, ***p < 0.001.</p

Image_2_Comparison and Functional Genetic Analysis of Striatal Protein Expression Among Diverse Inbred Mouse Strains.TIF

C57BL/6J (B6) and DBA/2J (D2) inbred mouse strains are highly variable genetically and differ in a large number of behavioral traits related to striatal function, including depression, anxiety, stress response, and response to drugs of abuse. The genetic basis of these phenotypic differences are, however, unknown. Here, we present a comparison of the striatal proteome between B6 and D2 and relate differences at the protein level to strain differences at the mRNA level. We also leverage a recombinant inbred BXD population derived from B6 and D2 strains to investigate the role of genetic variation on the regulation of mRNA and protein levels. Finally, we test the hypothesis that differential protein expression contributes to differential behavioral responses between the B6 and D2 strain. We detected the expression of over 2,500 proteins in membrane-enriched protein fractions from B6 and D2 striatum. Of these, 160 proteins demonstrated significant differential expression between B6 and D2 strains at a 10% false discovery level, including COMT, GABRA2, and cannabinoid receptor 1 (CNR1)—key proteins involved in synaptic transmission and behavioral response. Similar to previous reports, there was little overlap between protein and transcript levels (25%). However, the overlap was greater (51%) for proteins demonstrating genetic regulation of cognate gene expression. We also found that striatal proteins with significantly higher or lower relative expression in B6 and D2 were enriched for dopaminergic and glutamatergic synapses and processes involved in synaptic plasticity [e.g., long-term potentiation (LTP) and long-term depression (LTD)]. Finally, we validated higher expression of CNR1 in B6 striatum and demonstrated greater sensitivity of this strain to the locomotor inhibiting effects of the CNR1 agonist, Δ9-tetrahydrocannabinol (THC). Our study is the first comparison of differences in striatal proteins between the B6 and D2 strains and suggests that alterations in the striatal proteome may underlie strain differences in related behaviors, such as drug response. Furthermore, we propose that genetic variants that impact transcript levels are more likely to also exhibit differential expression at the protein level.</p

Table_3_Comparison and Functional Genetic Analysis of Striatal Protein Expression Among Diverse Inbred Mouse Strains.XLSX

C57BL/6J (B6) and DBA/2J (D2) inbred mouse strains are highly variable genetically and differ in a large number of behavioral traits related to striatal function, including depression, anxiety, stress response, and response to drugs of abuse. The genetic basis of these phenotypic differences are, however, unknown. Here, we present a comparison of the striatal proteome between B6 and D2 and relate differences at the protein level to strain differences at the mRNA level. We also leverage a recombinant inbred BXD population derived from B6 and D2 strains to investigate the role of genetic variation on the regulation of mRNA and protein levels. Finally, we test the hypothesis that differential protein expression contributes to differential behavioral responses between the B6 and D2 strain. We detected the expression of over 2,500 proteins in membrane-enriched protein fractions from B6 and D2 striatum. Of these, 160 proteins demonstrated significant differential expression between B6 and D2 strains at a 10% false discovery level, including COMT, GABRA2, and cannabinoid receptor 1 (CNR1)—key proteins involved in synaptic transmission and behavioral response. Similar to previous reports, there was little overlap between protein and transcript levels (25%). However, the overlap was greater (51%) for proteins demonstrating genetic regulation of cognate gene expression. We also found that striatal proteins with significantly higher or lower relative expression in B6 and D2 were enriched for dopaminergic and glutamatergic synapses and processes involved in synaptic plasticity [e.g., long-term potentiation (LTP) and long-term depression (LTD)]. Finally, we validated higher expression of CNR1 in B6 striatum and demonstrated greater sensitivity of this strain to the locomotor inhibiting effects of the CNR1 agonist, Δ9-tetrahydrocannabinol (THC). Our study is the first comparison of differences in striatal proteins between the B6 and D2 strains and suggests that alterations in the striatal proteome may underlie strain differences in related behaviors, such as drug response. Furthermore, we propose that genetic variants that impact transcript levels are more likely to also exhibit differential expression at the protein level.</p

Table_1_Comparison and Functional Genetic Analysis of Striatal Protein Expression Among Diverse Inbred Mouse Strains.XLSX

C57BL/6J (B6) and DBA/2J (D2) inbred mouse strains are highly variable genetically and differ in a large number of behavioral traits related to striatal function, including depression, anxiety, stress response, and response to drugs of abuse. The genetic basis of these phenotypic differences are, however, unknown. Here, we present a comparison of the striatal proteome between B6 and D2 and relate differences at the protein level to strain differences at the mRNA level. We also leverage a recombinant inbred BXD population derived from B6 and D2 strains to investigate the role of genetic variation on the regulation of mRNA and protein levels. Finally, we test the hypothesis that differential protein expression contributes to differential behavioral responses between the B6 and D2 strain. We detected the expression of over 2,500 proteins in membrane-enriched protein fractions from B6 and D2 striatum. Of these, 160 proteins demonstrated significant differential expression between B6 and D2 strains at a 10% false discovery level, including COMT, GABRA2, and cannabinoid receptor 1 (CNR1)—key proteins involved in synaptic transmission and behavioral response. Similar to previous reports, there was little overlap between protein and transcript levels (25%). However, the overlap was greater (51%) for proteins demonstrating genetic regulation of cognate gene expression. We also found that striatal proteins with significantly higher or lower relative expression in B6 and D2 were enriched for dopaminergic and glutamatergic synapses and processes involved in synaptic plasticity [e.g., long-term potentiation (LTP) and long-term depression (LTD)]. Finally, we validated higher expression of CNR1 in B6 striatum and demonstrated greater sensitivity of this strain to the locomotor inhibiting effects of the CNR1 agonist, Δ9-tetrahydrocannabinol (THC). Our study is the first comparison of differences in striatal proteins between the B6 and D2 strains and suggests that alterations in the striatal proteome may underlie strain differences in related behaviors, such as drug response. Furthermore, we propose that genetic variants that impact transcript levels are more likely to also exhibit differential expression at the protein level.</p

R script from calibration analysis.

(R)</p

Main tail withdrawal latency data file.

All data collected from our sample analgesic drug study and baseline latency comparison across strains. (CSV)</p

<i>TailTimer</i> detects acute changes in tail withdrawal latency following oral oxycodone exposure.

Pain thresholds of SD males (n = 8) were evaluated at 15 min, 1 h, and 4 h post-gavage of 3 mg/kg oxycodone or distilled water at a controlled water temperature of 48°C ± 0.25 and a fixed, low water mixing speed (setting 1). Latencies were significantly longer at 15 min and 1 h, but not 4 h, post-gavage of oxycodone versus distilled water. Following gavage of the distilled water control, latencies were not significantly different from baseline at any time point. Mean latencies reflect the average of the (two–four) measurements per individual rat averaged across timepoints. Data are expressed as mean ± SEM; ***p < 0.001.</p