Involvement of Noradrenergic Neurotransmission in the Stress- but not Cocaine-Induced Reinstatement of Extinguished Cocaine-Induced Conditioned Place Preference in Mice: Role for β-2 Adrenergic Receptors

The responsiveness of central noradrenergic systems to stressors and cocaine poses norepinephrine as a potential common mechanism through which drug re-exposure and stressful stimuli promote relapse. This study investigated the role of noradrenergic systems in the reinstatement of extinguished cocaine-induced conditioned place preference by cocaine and stress in male C57BL/6 mice. Cocaine- (15 mg/kg, i.p.) induced conditioned place preference was extinguished by repeated exposure to the apparatus in the absence of drug and reestablished by a cocaine challenge (15 mg/kg), exposure to a stressor (6-min forced swim (FS); 20–25°C water), or administration of the α-2 adrenergic receptor (AR) antagonists yohimbine (2 mg/kg, i.p.) or BRL44408 (5, 10 mg/kg, i.p.). To investigate the role of ARs, mice were administered the nonselective β-AR antagonist, propranolol (5, 10 mg/kg, i.p.), the α-1 AR antagonist, prazosin (1, 2 mg/kg, i.p.), or the α-2 AR agonist, clonidine (0.03, 0.3 mg/kg, i.p.) before reinstatement testing. Clonidine, prazosin, and propranolol failed to block cocaine-induced reinstatement. The low (0.03 mg/kg) but not high (0.3 mg/kg) clonidine dose fully blocked FS-induced reinstatement but not reinstatement by yohimbine. Propranolol, but not prazosin, blocked reinstatement by both yohimbine and FS, suggesting the involvement of β-ARs. The β-2 AR antagonist ICI-118551 (1 mg/kg, i.p.), but not the β-1 AR antagonist betaxolol (10 mg/kg, i.p.), also blocked FS-induced reinstatement. These findings suggest that stress-induced reinstatement requires noradrenergic signaling through β-2 ARs and that cocaine-induced reinstatement does not require AR activation, even though stimulation of central noradrenergic neurotransmission is sufficient to reinstate

Mechanosensory and ATP Release Deficits following Keratin14-Cre-Mediated TRPA1 Deletion Despite Absence of TRPA1 in Murine Keratinocytes.

Keratinocytes are the first cells that come into direct contact with external tactile stimuli; however, their role in touch transduction in vivo is not clear. The ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) is essential for some mechanically-gated currents in sensory neurons, amplifies mechanical responses after inflammation, and has been reported to be expressed in human and mouse skin. Other reports have not detected Trpa1 mRNA transcripts in human or mouse epidermis. Therefore, we set out to determine whether selective deletion of Trpa1 from keratinocytes would impact mechanosensation. We generated K14Cre-Trpa1fl/fl mice lacking TRPA1 in K14-expressing cells, including keratinocytes. Surprisingly, Trpa1 transcripts were very poorly detected in epidermis of these mice or in controls, and detection was minimal enough to preclude observation of Trpa1 mRNA knockdown in the K14Cre-Trpa1fl/fl mice. Unexpectedly, these K14Cre-Trpa1fl/fl mice nonetheless exhibited a pronounced deficit in mechanosensitivity at the behavioral and primary afferent levels, and decreased mechanically-evoked ATP release from skin. Overall, while these data suggest that the intended targeted deletion of Trpa1 from keratin 14-expressing cells of the epidermis induces functional deficits in mechanotransduction and ATP release, these deficits are in fact likely due to factors other than reduction of Trpa1 expression in adult mouse keratinocytes because they express very little, if any, Trpa1

<i>Trpa1</i> mRNA is poorly detected in mouse epidermal keratinocytes.

<p>(<b>A</b>) Following Cre-mediated recombination of genomic <i>Trpa1</i> DNA, an excision product is amplified in the epidermis of <i>K14Cre</i>-<i>Trpa1</i>^fl/fl mice (left), but not in the control (<i>K14Cre-Trpa1</i>^<i>+/+</i>) mice. Mutant mice (<i>K14Cre-Trpa1</i>^<i>fl/fl</i>) mice were either from an independently maintained colony (mutant-colony), or generated as littermates from heterozygous breeders (mutant—littermate). No excision product was observed in the DRGs of either control or mutant mice. Positive control band was obtained from DRG tissue from an <i>AdvillinCre-Trpa1</i>^<i>fl/fl</i> mouse (right). For panels B-F, mRNA was isolated from DRG and epidermis from control mice. (<b>B</b>) Amplification plot showing <i>Gapdh</i> and <i>Trpa1</i> mRNA transcript amplification. <i>Gapdh</i> was consistently detected and quantified in both DRG and epidermal samples (top). <i>Trpa1</i> mRNA was strongly detected in DRG samples; however, the same PCR protocol did not detect measurable <i>Trpa1</i> in epidermal samples (bottom). (<b>C</b>) Two different primer sets to detect <i>Trpa1</i> were effective in measuring <i>Trpa1</i> from DRG samples using SYBR Green qPCR, but did not amplify <i>Trpa1</i> from epidermal samples or cultured epidermal keratinocytes. Primer set 1 targets exons 22–23 within the deleted pore region of <i>Trpa1</i>; primer set 2 targets exons 17–19, upstream of the deleted pore region. (<b>D</b>) Three sets of Taqman primer-probes were similarly unable to detect <i>Trpa1</i> transcripts in control epidermis. Primer set 3 targets exons 13–14, set 4 targets exons 22–23, and set 5 targets exons 23–24 of <i>Trpa1</i>. (<b>E</b>) Neither qPCR for exons 22–23 (set 1) nor exons 22–23 (set 4) were capable of detecting <i>Trpa1</i> in neonatal mouse epidermis. (<b>F</b>) Two days after hindpaw injection of CFA, <i>Trpa1</i> remained undetected in epidermis. <i>N</i>.<i>d</i>. denotes transcript not detected.</p

No <i>Trpa1</i> knockdown is observed in epidermis of mutant (<i>K14Cre-Trpa1</i>^<i>fl/fl</i>) mice compared to controls (<i>K14Cre-Trpa1</i>^<i>+/+</i>).

<p>(<b>A</b>) Gene-specific primers to improve reverse transcription of <i>Trpa1</i> were used prior to targeted amplification of <i>Trpa1</i>. This sensitive method detected a very small amount of <i>Trpa1</i> transcripts within hindpaw epidermis, and the amplification was equivalent between control and mutant samples. (<b>B-D</b>) Quantitative digital-droplet PCR was performed on samples isolated from epidermis of control and mutant mice. (<b>B</b>) TRPV3 was efficiently detected in all samples tested. No template control (NTC) bars denote ddPCR performed with no cDNA added. (<b>C</b>) ddPCR for exons 23–24 of <i>Trpa1</i> (within the loxp-flanked region) detected ample transcripts within the DRG samples. On average, less than one positive event per entire reaction volume was detected in both control and mutant samples. A control sample of DRG tissue from an <i>AdvillinCre-Trpa1</i>^fl/fl mouse shows that Cre-mediated recombination in a different tissue (sensory neurons) reduces <i>Trpa1</i> mRNA detection (DRG_Adv). (<b>D</b>) ddPCR for exons 12–13 detected on average less than one positive event per epidermal sample. <i>N</i>.<i>d</i>. denotes transcript not detected. **** <i>P<</i>0.0001, compared to every other bar.</p

Mutant mice exhibit minimal inflammatory allodynia and hyperalgesia two days after CFA injection.

<p>(<b>A</b>) Following injection of CFA into the hindpaw, control mice exhibited a marked increase in mechanical sensitivity following CFA injection into the hindpaw as indicated by the reduction in paw withdrawal threshold. In contrast, no changes in threshold were observed in mutant mice treated with CFA. After inflammation, there remained a striking difference in paw withdrawal threshold between mutant and control animals. Analysis was performed using a 2-way ANOVA with <i>post hoc</i> analysis via Mann-Whitney <i>U</i> tests with Bonferroni adjustment. (<b>B</b>) As indicated by a significant increase in paw withdrawal frequency to repeated application of a 3.31 mN force (left), control mice displayed hypersensitivity after CFA injection. Mutant mice exhibited a smaller, yet significant, increase in withdrawal frequency (right). (<b>C</b>) CFA injection induced an increase in the injected paw thickness of both control and mutant mice. Following CFA, control paws were significantly thicker that mutant paws, though the effect size was limited. (<b>D</b>) Similarly, CFA increased paw width in both control and mutant mice. (<b>E</b>) There were no overall differences in paw withdrawal latency to a 0°C cold plate, though there was a strong trend suggesting CFA may have decreased cold withdrawal latency only in the control mice. (<b>F</b>) CFA greatly increased the <i>number</i> of paw lifts and responses to a 0°C cold plate in control mice, and did not significantly increase paw responses in mutant mice. * <i>P</i><0.05, ** <i>P</i><0.01; *** <i>P</i><0.001, **** <i>P</i><0.0001.</p

Small-diameter sensory neurons from mutant mice have relatively few deficits in chemical activation.

<p>(<b>A</b>) Calcium imaging of small-diameter DRG neurons revealed no difference in the percentage of neurons responding to the 100 μM of the TRPA1 agonist, cinnamaldehyde, when comparing control and mutant neurons. (<b>B</b>) Magnitude response to cinnamaldehyde was similar between naïve control and mutant small-diameter neurons. (<b>C-D</b>) A similar proportion of control and mutant small-diameter neurons responded to another TRPA1 agonist, mustard oil (100 μM; 3 mice per genotype). However, there was a subtle decrease in the magnitude calcium response in mutant compared to control neurons. (<b>E-F</b>) Control and mutant sensory neurons responded similarly to application of acidic solutions of pH 6.0 (E) or pH 5.0 (F). (<b>G</b>) Combining across multiple experiments, the magnitude response to a depolarizing stimulus of 50 mM K⁺ was similar between control and mutant small-diameter neurons. ** <i>P</i><0.01.</p

Mutant mice (<i>K14Cre-Trpa1</i>^fl/fl) exhibit decreased behavioral sensitivity to noxious and gentle mechanical stimuli.

<p>(<b>A</b>) Paw withdrawal responses revealed markedly elevated thresholds in mutant mice. (<b>B</b>) Mutant mice had significantly fewer responses to repeated application of a 3.31 mN von Frey filament. (<b>C</b>) Mutant mice responded less frequently to repeated applications of a spinal needle. (<b>D</b>). Mutant mice responded less frequently to repeated, punctate application of a 0.7 mN von Frey filament to the plantar hindpaw. (<b>E</b>) Mutant mice exhibited decreased responses to gentle stroking puffed-out cotton swab applied to the hindpaw. * <i>P</i><0.05; *** <i>P</i><0.001, **** <i>P<</i>0.0001. Data reported as mean ± s.e.m.</p

Cre recombinase activity was visualized in epidermal tissues of <i>K14Cre-tdTomato</i> reporter mice.

<p>(<b>A-B</b>) tdTomato reporter fluorescence was observed in the epidermis of both glabrous and hairy skin sections, as expected. In glabrous skin, reporter fluorescence was also observed in sebaceous glands (arrowhead). Bottom row presents the lack of fluorescence in the <i>tdTomato</i>^LSL mice in the absence of the <i>K14Cre</i> allele. (<b>C</b>) No tdTomato reporter fluorescence was detected in the DRG of either control or mutant animals, suggesting no ectopic Cre recombinase activity was present in these sensory neurons. (<b>D</b>) Importantly, in the absence of the <i>K14Cre</i> allele, the <i>Trpa1</i>^fl/fl mice did not show any mechanical sensory deficit compared to wildtype C57BL/6 mice. (<b>E</b>) Mutant animals from the same litters as controls exhibited significant elevated mechanical thresholds (littermate mutants vs. littermate controls). Furthermore, although there was a trend for the mutant animals from the independent colony to have an even greater mechanosensitivity deficit, their mechanical thresholds were not statistically different from those of littermate mutant animals (<i>p</i> = 0.0503). Analysis was performed via Kruskall-Wallis and a Dunn’s <i>post hoc</i> test. *** <i>P</i>< 0.001, **** <i>P</i>< 0.0001.</p