PICK1-positive cells were observed in the DRG of WT mice, but not of PICK1 KO mice (Figure ). Most were small, ranging from 250 to 750 μm2
in surface area (Figure ). Some medium-size DRG cells were also PICK1-positive (Figure ). In contrast to small DRG cells, the population of large-diameter DRG cells mostly lacked PICK1. Approximately 29.7% of the 1124 DRG cells counted were PICK1-positive. Double-labeling studies showed that PICK1 was colocalized exclusively with NeuN, a marker for neuronal nuclei [21
], but was absent from glia (Figure ). We further identified the cytochemical characteristics of PICK1-positive neurons in the DRG. Approximately 62.8% (211/336) of PICK1-positive neurons were positive for IB4, a marker for small DRG non-peptidergic neurons [21
], and approximately 26.9% (125/465) of PICK1-positive neurons were positive for CGRP, a marker for small DRG peptidergic neurons [21
Figure 1 Localization and distribution of PICK1 immunoreactivity in the dorsal root ganglion (DRG). A, Distribution of PICK1 immunoreactivity in the DRG cells of wild-type (WT; left) and PICK1 knockout (KO, right) mice. No immunoreactivity for PICK1 was observed (more ...)
Figure 2 PICK1 immunoreactivity in the DRG neurons and colocalization with IB4 binding and CGRP. Digital images were taken with a confocal laser-scanning microscope. A, Colocalization of PICK1 and NeuN. B, Colocalization of PICK1 and IB4 binding. C, Colocalization (more ...)
PICK1 protein was also observed in the spinal cord. PICK1 immunoreactivity was distributed at a higher density in the superficial dorsal horn than in other regions of spinal cord, particularly in the lamina I and inner lamina II (Figure ). Under high magnification, immunoreactivity to PICK1 was observed mostly as punctate or patch immunostaining (Figure ). A few PICK1-positive cell bodies were seen in dorsal horn (Figure ). Under electron microcopy, immunogold-labeling for PICK1 was prominent in the postsynaptic density, dendrites, and presynaptic terminals in the superficial dorsal horn (Figure and ).
Figure 3 Localization and distribution of PICK1 immunoreactivity in the spinal cord. A, PICK1 immunoreactivity in the dorsal horn was strongest in lamina I and inner lamina II (IIi). IIo: outer lamina II. B, High magnification of the outlined region from A. PICK1-positive (more ...)
We further used behavioral testing to determine whether PICK1 participated in transmission and modulation of nociceptive information. Because PICK1 is a scaffolding protein without any receptor-like or enzyme-like activities, we used a genomic strategy in which the PICK1 gene was mutated. As reported previously [16
], PICK1 KO male and female mice are viable with normal appearance. The genomic status of each mouse was checked with PCR (Figure ), and the expression of PICK1 protein in the DRG and spinal cord also was verified by Western blotting analysis and immunohistochemistry. As expected, PICK1 was not detected in the DRG or spinal cord of the PICK1 KO mice (Figures and ). In addition, the expression of PICK1 interacting proteins was evaluated in the DRG and spinal cord. Western blotting analysis revealed that the levels of interacting proteins ASIC1 and ASIC2 in the DRG (Figure ) and the levels of interacting proteins GluR2 and PKCα in the spinal cord (Figure ) were normal in the PICK1 KO mice. The DRG and spinal cord cell architecture appeared normal in PICK1 KO mice (data not shown). These results suggest that the targeted disruption of PICK1 gene does not produce any detectable abnormality of DRG or spinal cord anatomical structure or protein expression except for the loss of PICK1 protein.
Figure 4 Biochemical characterization of PICK1 knockout (KO) mice. A, PCR analysis of tail DNA from wild-type (WT), heterozygous (HET), and PICK1 KO mice. M: molecular weight standard. B, Western blot analysis shows the expression of PICK1 protein and its interacting (more ...)
We first examined whether the deletion of PICK1 affected acute nociception. Acute mechanical sensitivity was assessed by response frequencies of paw withdrawal to mechanical stimulation elicited by different forces of von Frey filaments. Acute thermal sensitivity was evaluated by high-intensity radiant heat applied to the plantar sides of left and right hind paws and the tail. We found that paw withdrawal responses of PICK1 KO mice to acute mechanical (Figure ) and thermal stimuli (Figure ) as well as tail flick response to acute thermal stimulation (Figure ) were similar to those of WT mice. These data indicate that transmission of acute pain messages is preserved in PICK1 KO mice.
Figure 5 PICK1 knockout (KO) preserves acute pain. A, Baseline paw withdrawal frequencies in response to various forces of von Frey monofilaments in naïve wild-type (WT) and PICK1 KO mice. B, Baseline paw withdrawal latencies in response to heat stimulation (more ...)
Next, we determined the role of PICK1 in neuropathic pain. Consistent with our previous studies [6
SNL produced long-term mechanical and thermal pain hypersensitivity on the hind paw ipsilateral to nerve injury in WT mice. The application of 0.24 mN (low intensity) and 4.33 mN (moderate intensity) von Frey filaments to the plantar side of the ipsilateral hind paw elicited paw withdrawal frequencies that were significantly greater than those at pre-injury baseline values. This mechanical pain hypersensitivity was detectable on day 3, reached a peak level between days 7 and 9, and persisted for at least 14 days post-nerve injury (Figure and ). Likewise, paw withdrawal latencies in response to application of heat to the plantar side of the ipsilateral hind paw were markedly decreased from the pre-injury baseline values. This thermal pain hypersensitivity developed within 5 days and persisted for at least 14 days (Figure ). The baseline withdrawal responses to mechanical and thermal stimuli were similar in WT and PICK1 KO mice as described above (Figure ), but PICK1 KO mice did not display mechanical or thermal pain hypersensitivity following spinal nerve injury. Paw withdrawal responses to mechanical and thermal stimuli were unchanged compared to the baseline levels in the PICK1 KO mice (P
> 0.05; Figure ). As expected, paw withdrawal responses to mechanical and thermal stimuli were not altered in the contralateral hind paw in either WT or PICK1 KO mice after spinal nerve injury (Figure ).
Figure 6 PICK1 knockout (KO) mice exhibit impaired neuropathic pain. L5 SNL significantly increased paw withdrawal frequencies in response to 0.24 mN (low intensity; A) and 4.33 mN (moderate intensity; B) mechanical stimuli and significantly decreased paw withdrawal (more ...)
Because locomotor function is involved in paw withdrawal reflex response, we tested whether the deletion of PICK1 affected locomotor functions. We recorded the scores for three gross reflexes (placing, grasping, and righting) in WT and PICK1 KO mice. We found that PICK1 KO mice performed the same as their WT littermates for the three reflexes (Table ) and did not exhibit gross motor coordination defects. The results suggest that gross locomotor function is not impaired by PICK1 deficiency and cannot account for the observed deficiency of nerve injury-induced pain behaviors.
Mean (SEM) changes in locomotor test
PICK1 genetic KO mice, like other congenital KO mice, might undergo unknown compensatory changes in the nervous system during development. Such changes might affect animal behaviors. To further confirm the role of PICK1 in neuropathic pain, we used PICK1 AS ODN to acutely and transiently knock down PICK1 in spinal cord and DRG of rats. Our previous study showed that i.th. administration of PICK1 AS ODN (10 μg), but not MS ODN (10 μg), selectively and significantly reduced PICK1 expression in spinal cord and DRG [14
]. This reduction did not affect basal mechanical and thermal responses or locomotor function, but it did attenuate CFA-induced mechanical and thermal pain hypersensitivities [14
]. Here, we found that after i.th. injection of 10 μg PICK1 AS ODN, mechanical and thermal pain hypersensitivities were markedly blunted until day 7 post-SNL (Figure ). Significant differences in paw withdrawal frequencies and latencies were observed between the AS group (n = 5) and saline group (n = 5; P
< 0.01). As expected, 10 μg of MS ODN did not affect the development of nerve injury-induced mechanical and thermal pain hypersensitivities (P
> 0.05, n = 5; Figure ).
Figure 7 PICK1 knockdown blunts neuropathic pain. L5 spinal nerve ligation (SNL) led to a significant increase in paw withdrawal frequency in response to 8.01 mN mechanical stimulation (A) and a marked decrease in paw withdrawal latency in response to heat stimulation (more ...)
Results from our previous study suggested that PICK1-mediated dorsal horn GluR2 internalization contributes to the maintenance of CFA-induced chronic inflammatory pain. Therefore, we examined whether a similar process might occur in neuropathic pain. Crude cytosolic and synaptosomal membrane protein fractions were separated by using differential centrifugation. Consistent with our previous studies [11
], a plasma membrane-specific protein, N
-cadherin, was detected highly in the membrane fractions, but not in the cytosolic fraction (data not shown), indicating that the fractionation procedure effectively separated cytosolic proteins from synaptosomal membrane proteins. We found that L5
SNL did not change the level of GluR2 in either the cytosolic or the synaptosomal fraction from the ipsilateral L4
(data not shown) and L5
(Figure ) dorsal horns of WT and PICK1 KO mice during the observation period. These findings indicate that peripheral nerve injury does not induce GluR2 internalization in dorsal horn.
Figure 8 L5 spinal nerve ligation (SNL) does not induce GluR2 internalization in dorsal horns from wild-type (WT) mice. A, GluR2 levels in the cytosolic fraction from the ipsilateral dorsal horns of WT and PICK1 knockout (KO) mice at different time points after (more ...)