Conditional disruption of calpain in the CNS alters dendrite morphology, impairs LTP, and promotes neuronal survival following injury.

Ubiquitous classical (typical) calpains, calpain-1 and calpain-2, are Ca(+2)-dependent cysteine proteases, which have been associated with numerous physiological and pathological cellular functions. However, a clear understanding of the role of calpains in the CNS has been hampered by the lack of appropriate deletion paradigms in the brain. In this study, we describe a unique model of conditional deletion of both calpain-1 and calpain-2 activities in mouse brain, which more definitively assesses the role of these ubiquitous proteases in brain development/function and pathology. Surprisingly, we show that these calpains are not critical for gross CNS development. However, calpain-1/calpain-2 loss leads to reduced dendritic branching complexity and spine density deficits associated with major deterioration in hippocampal long-term potentiation and spatial memory. Moreover, calpain-1/calpain-2-deficient neurons were significantly resistant to injury induced by excitotoxic stress or mitochondrial toxicity. Examination of downstream target showed that the conversion of the Cdk5 activator, p35, to pathogenic p25 form, occurred only in the presence of calpain and that it played a major role in calpain-mediated neuronal death. These findings unequivocally establish two central roles of calpain-1/calpain-2 in CNS function in plasticity and neuronal death.

Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment.

Mutations in phosphatase and tensin homologue-induced kinase 1 (PINK1) cause recessively inherited Parkinson's disease (PD), a neurodegenerative disorder linked to mitochondrial dysfunction. In healthy mitochondria, PINK1 is rapidly degraded in a process involving both mitochondrial proteases and the proteasome. However, when mitochondrial import is compromised by depolarization, PINK1 accumulates on the mitochondrial surface where it recruits the PD-linked E3 ubiquitin ligase Parkin from the cytosol, which in turn mediates the autophagic destruction of the dysfunctional organelles. Using an unbiased RNA-mediated interference (RNAi)-based screen, we identified four mitochondrial proteases, mitochondrial processing peptidase (MPP), presenilin-associated rhomboid-like protease (PARL), m-AAA and ClpXP, involved in PINK1 degradation. We find that PINK1 turnover is particularly sensitive to even modest reductions in MPP levels. Moreover, PINK1 cleavage by MPP is coupled to import such that reducing MPP activity induces PINK1 accumulation at the mitochondrial surface, leading to Parkin recruitment and mitophagy. These results highlight a new role for MPP in PINK1 import and mitochondrial quality control via the PINK1­Parkin pathway.

Metabotropic glutamate receptors modulate glutamatergic and GABAergic synaptic transmission in the central nucleus of the inferior colliculus.

Fast glutamatergic and GABAergic transmission in the central nucleus of the inferior colliculus (ICC), a major auditory midbrain structure, is mediated respectively by alpha-amino-3-hydroxy-5-methylisoxazole-4 propionic acid (AMPA) and gamma-aminobutyric acid (GABA)(A) receptors. In this study, we used whole-cell patch clamp recordings in brain slices to investigate the effects of activation of metabotropic glutamate receptors (mGluRs) on synaptic responses mediated by AMPA and GABA(A) receptors in ICC neurons of young rats. Excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) mediated respectively by AMPA and GABA(A) receptors were elicited by stimulation of the lateral lemniscus, the major afferent pathway to the ICC. The agonists for groups I and II mGluRs, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD), and for group III mGluRs, L-2-amino-3-hydroxypropanoic acid 3-phosphate (L-SOP), did not affect intrinsic membrane properties of the ICC neurons. The agonist for group II mGluRs, (1R,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0] hexane-4,6-dicarboxylic acid (LY379268), significantly reduced the AMPA receptor-mediated EPSCs and GABA(A) receptor-mediated IPSCs. The effects were reversed by the group II mGluR antagonist, (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495). The agonists for groups I and III, (RS)-3,5-dihydroxyphenylglycine (DHPG) and L-SOP, respectively, did not affect AMPA or GABA(A) receptor-mediated responses. The reduction of the synaptic responses by LY379268 was accompanied by a substantial increase in a ratio of the second to the first AMPA receptor-mediated EPSCs and GABA(A) receptor-mediated IPSCs to paired-pulse stimulation. The results suggest that group II mGluRs regulate both fast glutamatergic and GABAergic synaptic transmission in the ICC, probably through a presynaptic mechanism due to reduction of transmitter release.

Single whisker experience started on postnatal days 0, 5 or 8 changes temporal characteristics of response integration in layers IV and V of rat barrel cortex neurons.

Neonatal single whisker experience changes the response properties of spared barrel neurons to deflections of principal and adjacent whiskers. However little is known about the temporal characteristics of the paired whisker inputs. To address this issue we used computer controlled mechanical displacement of paired whiskers in control and plucked animals (plucking of all whiskers but D2 started at 0, 5 and 8 postnatal days). The principal whisker (PW) and its caudal adjacent whisker (AW) were deflected simultaneously or serially at different inter-stimulus intervals (10, 20, 30, 50 and 100 ms). Neuronal responses were recorded in D2 spared barrel both in layers IV and V. In the control group, combined deflection of AW prior to PW led to suppression of ON and OFF responses to PW deflection both in layers IV and V. The magnitude of this suppression was strongly dependent on the inter-stimulus intervals (ISIs). At almost all tested ISIs, whisker plucking from P0, P5 and P8 weakened suppressive interactions in layers IV and V barrel neurons for both ON and OFF responses. The most decrease in inhibitory interactions was observed in P5 plucked animals. Principal whisker-evoked ON responses were increased only in P0 plucked animals both in layers IV and V. AW-evoked ON responses are decreased in P5 plucked animals in layer IV. The available data suggest that sensory experience can modulate temporal aspects of response integration and receptive field properties of layers IV and V neurons in barrel cortex. These changes have different critical periods.

Electrical stimulation of locus coeruleus strengthens the surround inhibition in layer V barrel cortex in rat.

It is believed that locus coeruleus (LC) influences the sensory information processing. However, its role in cortical surround inhibitory mechanism is not well established. In this experiment, using controlled mechanical displacement of whiskers; we investigated the effect of electrical stimulation of LC on response of layer V barrel cortical neurons in anesthetized rat. LC was stimulated 0, 50, 100, 200 and 400 ms before principal or adjacent whiskers deflection. For assessing the effect of LC stimulation on inhibitory receptive filed of barrel neurons, adjacent whisker was also deflected 20 ms before principal whisker deflection, and LC stimulation was applied 0-400 ms before principal whisker displacement. We found that LC stimulation increase the response magnitude of layer V neurons to principal whisker deflection (significant in 50-400 ms intervals). This increase was also observed in response to adjacent whisker deflection (significant in 100 ms interval). The response latency of neurons was decreased when LC was stimulated 400 ms before principal whisker deflection but LC stimulation did not affect the neuronal response latency to adjacent whisker displacement. Inhibitory effect of adjacent whisker deflection on neuronal response magnitude was increased by LC stimulation, tested in combined whisker displacement. These findings suggest that LC, by modulating the neuronal responses, enhances the neuronal responsiveness to sensory stimuli and increases their surround inhibition in cortex.

Dorsal raphe nucleus stimulation modulates the response of layers IV and V barrel cortical neurons in rat.

The effect of dorsal raphe nucleus (DRN) electrical stimulation on response properties of layers IV and V barrel cortical neurons was studied. To assess the receptive field characteristics of cortical neurons, responses of neurons were recorded following the displacement of principal and adjacent whiskers individually or in a condition test paradigm. Then neuronal responses to the displacement of whiskers were analyzed following DRN stimulation at 0, 50, 100, 200 and 400 ms inter-stimulation intervals. Considering On responses, DRN stimulation suppressed the response magnitude of layer V neurons to principal whisker deflection, while it slightly increased that of layer IV neurons (not statistically significant). The response latency of layer IV neurons increased when DRN was stimulated 200 or 400 ms before principal whisker deflection, while the response latency of layer V was not changed. DRN stimulation had no effect on either magnitude or latency of neuronal response to the adjacent whisker deflections. We observed a decrease in the inhibitory effect of the adjacent whisker deflection on the magnitude of neuronal response to the principal whisker deflection in layer IV when DRN was stimulated 200 ms before the principal whisker deflection. Off responses did not show any significant effect of DRN stimulation. Our results suggest a modulating role for DRN in processing of the incoming information into barrel cortex. This effect might be location dependent.

Eye-wiping test: a sensitive animal model for acute trigeminal pain studies.

The possibility of introducing eye-wiping test as a model of acute pain was examined in rat, and it was compared with the well-known hot plate test. One drop of NaCl 5 M was placed into the animal eye, and the number of eye wipes with the ipsilateral forelimb was counted during 30 s. The withdrawal latency in hot plate test was also examined. Afterward, animals were treated with morphine (1, 2, 4, 6, 8 or 10 mg/kg), imipramine (25 mg/kg), sodium salicylate (250 mg/kg) or saline (i.p). After 30 min, the animals were tested again with eye-wiping and hot plate tests. Our results showed that morphine injection dose dependently decreased the number of eye wipes and increased the response latency to hot plate tests. There was a good correlation between the analgesic effects of morphine on responses to both tests, however, morphine produced more pain relief in eye-wiping test. Imipramine significantly decreased the number of eye wipes and increased the response latency to hot plate test, while sodium salicylate and saline injection did not. It may be concluded that the eye-wiping test can be used as a reliable method in trigeminal pain studies, which is sensitive to opioid and tricyclic antidepressant in rat.

Effects of GABAA receptor inhibition on response properties of barrel cortical neurons in C-fiber-depleted rats.

C-fiber depletion results in expansion of low threshold somatosensory mechanoreceptive fields. In this study, we investigated the role of intact C-fibers in GABAA-mediated inhibition in barrel cortical neurons. We used electronically controlled mechanical stimulation of whiskers to quantitatively examine the responses of barrel cells to whisker displacements. After systemic injection of picrotoxin neuronal responses were recorded at 5 min intervals for 20 min and then at 10 min intervals for 100 min. Picrotoxin injection caused a 3-fold increase in response magnitude of adjacent whisker stimulation and 1.4-fold increase in response magnitude of principal whisker stimulation with a maximum enhancement 50 min after the injection. There was no significant change in spontaneous activity following picrotoxin injection. The response enhancement and receptive field expansion observed in normal rats were completely absent in the C-fiber-depleted rats. These results suggest that the GABAA-mediated inhibition that modulates the receptive field functional organization of the barrel cortex depends on intact C-fibers.

Effects of neonatal C-fiber depletion on discrimination of principal and adjacent whisker stimulation within rat individual cortical barrels.

Controlled mechanical displacement was used to stimulate single whiskers in normal and C-fiber depleted rats to quantitatively examine the role of C-fibers in the response properties of barrel cortical cells. C-fiber depletion using neonatal capsaicin treatment increased the barrel single-unit response magnitude to deflection of both principal and adjacent whiskers while there was not any significant difference in the barrel cells' spontaneous activity. Capsaicin treatment increased the neural response duration of adjacent whisker stimulation but did not change that to the principal whisker deflection. There was no difference in response latencies of principal or adjacent whisker displacement between the normal and C-fiber-depleted groups. The efficiency of neural code for differentiation of principal and adjacent whiskers was measured by ROC analysis, which reflects the performance of an ideal observer in this discrimination using cells' firing rate. No significant difference was found in the performance of neurons in capsaicin-treated and control groups in distinguishing principal and adjacent whisker deflections from each other. These results suggest that neonatal C-fiber depletion causes an expansion of barrel cells receptive field but it does not affect the discrimination of individual whisker stimulation by the barrel cells.