Ligation of mouse L4 and L5 spinal nerves produces robust allodynia without major motor function deficit.

Spinal nerve L5/L6 ligation (SNL) in rats has become the standard for mechanistic studies of peripheral neuropathy and screening for novel analgesics. Conventional SNL in our hybrid mice resulted in a wide range of allodynia. Anatomical evaluation indicated that a variable number of lumbar vertebrae existed, resulting in L4/L5 or L5/L6 being ligated. Surprisingly, L4/L5 ligation did not result in ipsilateral hind limb paralysis and produced robust allodynia. Following a recent report that the mouse L4 neural segment is homologous with rat L5 we generated L4, L5 or both L4 and L5 (L4/L5) ligations in C57 mice after establishing a modified set of surgical landmarks. In contrast to rats, L4 ligation in these mice did not result in hind limb paralysis. Robust allodynia was observed in all three ligation groups. Nerve degeneration confirmed that L4 and L5, respectively, are primary contributors to the tibial and sural branches of the sciatic nerve in mice. A larger von Frey sensitive area reflected the wider distribution of Wallerian degeneration in the hindlimb of L4- compared to L5-ligated mice. Ligation of mouse L4 and L5 spinal nerves produces consistent, robust neuropathic pain behaviors and is suitable as a model for investigating mechanisms of neuropathic pain and for testing of novel analgesics. Gabapentin, used as a validation drug in neuropathic pain models and as a reference compound for novel analgesics, significantly reduced allodynia in the mice tested (L4/L5 ligations). Given the ease of surgery, robust allodynia, and larger von Frey sensitive area, we conclude that combined ligation of spinal nerves L4 and L5 optimizes the SNL model in mice.

Characterization of PTPRG in knockdown and phosphatase-inactive mutant mice and substrate trapping analysis of PTPRG in mammalian cells.

Receptor tyrosine phosphatase gamma (PTPRG, or RPTPγ) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98-99% reduction of RPTPγ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPγ C1060S, replaced the wild type. We showed that human wild type RPTPγ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPγ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPγ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPγ for use in an assay for identifying inhibitors, "substrate trapping" mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPγs retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPγ did not. This suggested that wild type RPTPγ might auto-dephosphorylate which was confirmed by an in vitro dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPγ. Additional investigation of RPTPγ activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.

Differential sensitivity to SSRI and tricyclic antidepressants in juvenile and adult mice of three strains.

Clinical studies have shown differential efficacy of several antidepressants in children and adolescents compared to adults, yet few animal studies have sought to characterize this phenomenon. We compared effects of fluoxetine and imipramine in two common behavioral assays that hold high predictive validity for antidepressant activity, tail suspension and forced swim test, using juvenile (5 weeks) and adult (12 weeks) mice from 3 strains. C57BL/6J-Tyr(c-Brd) (C57), hybrid C57BL/6J-Tyr(c-Brd)x129S5/SvEvBrd (F2), and Balb/cAnNTac (Balb/C) mice were tested in forced swim test and tail suspension after i.p. dosing with either fluoxetine or imipramine. Brain tissues were analyzed to evaluate levels of VMAT2, a possible modulator of age-dependent sensitivity to antidepressants. Imipramine had more consistent antidepressant effect across age groups and strains. Imipramine increased struggle in mice of both ages. Fluoxetine did not have an effect on immobility in Balb/C of both ages in tail suspension. Fluoxetine also did not increase forced swim struggle behavior in juvenile mice of all strains, but was effective in increasing struggle in adults. Juvenile mice had higher immobility and lower struggle than adults in forced swim, and juveniles also had higher immobility in tail suspension test for Balb/C and C57. In addition, VMAT2 levels were increased in juveniles. These results confirm that standard antidepressants produce effects in both juveniles and adults but age-related differences were evident in both tests. Further examination of these effects is needed to determine whether it may be related to age-dependent difference in the clinical response to antidepressants of these classes.

Learning and memory impairment in Eph receptor A6 knockout mice.

Genetic inhibition of the ephrin receptor (EphA6) in mice produced behavioral deficits specifically in tests of learning and memory. Using a fear conditioning training paradigm, mice deficient in EphA6 did not acquire the task as strongly as did wild type (WT) mice. When tested in the same context 24h later, knockout (KO) mice did not freeze as much as WT mice indicating reduced memory of the consequences of the training context. The KO mice also displayed less freezing when presented with the conditioning stimulus (CS) in a separate context. In the hidden platform phase of the Morris water maze (MWM) task, KO mice did not reach the same level of proficiency as did WT mice. KO mice also exhibited less preference for the target quadrant during a probe trial and were significantly impaired on an initial reversal of the platform. These findings suggest that EphA6, in line with a number of other Eph receptors and their ephrin ligands, is involved in neural circuits underlying aspects of learning and memory.

Amygdalar lateralization in fear conditioning: evidence for greater involvement of the right amygdala.

The relative contribution of left and right amygdalae in the acquisition and retention of fear conditioning was investigated in rats. Pretraining bilateral electrolytic lesions blocked the acquisition of conditioned fear to tone and context, whereas unilateral lesions induced partial impairments with no left-right amygdala differences. In contrast, posttraining bilateral and unilateral lesions produced significant deficits in the retention of conditioned fear to tone and context. Although no left-right difference was observed to tone, the right amygdala lesions generated greater deficits in contextual fear than the left amygdala lesions. These results indicate that fear conditioning is partially disrupted with unilateral amygdalar lesions, but that the right amygdala has greater involvement than the left amygdala when conditioning occurs under a normal brain state.

Effects of stress and hippocampal NMDA receptor antagonism on recognition memory in rats.

Exposures to uncontrollable stress have been shown to alter ensuing synaptic plasticity in the hippocampus and interfere with hippocampal-dependent spatial memory in rats. The present study examined whether stress, which impairs hippocampal long-term potentiation (LTP), also affects (nonspatial) hippocampal-dependent object-recognition memory, as tested on the visual paired comparison task (VPC) in rats. After undergoing an inescapable restraint-tailshock stress experience, rats exhibited markedly impaired recognition memory at the 3-h (long) familiarization-to-test phase delay but not at the 5-min (short) delay. In contrast, unstressed control animals showed robust recognition memory (i.e., they exhibited reliable preferences for novel over familiar objects) at both short- and long-delay periods. The impairing effect of stress on long-delay recognition memory was transient because 48 h after undergoing stress experience, animals performed normally at the long delay. Similar to stress, microinfusions of DL-2-amino-5-phosphonovaleric acid (APV), a competitive N-methyl-D-aspartate receptor (NMDAR) antagonist that blocks LTP, into the dorsal hippocampus selectively impaired object-recognition memory at the long-delay period. Together, these results suggest that stress and intrahippocampal administration of APV affect recognition memory by influencing synaptic plasticity in the hippocampus.