Aging causes a slowing in ciliary beat frequency, mediated by PKCε.

The elderly are at much higher risk for developing pneumonia than younger individuals. Pneumonia is a leading cause of death and is the third most common reason for hospitalization in the elderly. One reason that elderly people may be more susceptible to pneumonia is a breakdown in the lung's first line of defense, mucociliary clearance. Cilia beat in a coordinated manner to propel out invading microorganisms and particles. Ciliary beat frequency (CBF) is known to slow with aging, however, little is known about the mechanism(s) involved. We compared the CBF in BALB/c and C57BL/6 mice aged 2, 12, and 24 mo and found that CBF diminishes with age. Cilia in the mice at age 12 and 24 mo retained their ability to be stimulated by the ß2 agonist procaterol. To help determine the mechanism of ciliary slowing, we measured protein kinase C alpha and epsilon (PKCα and PKCε) activity. There were no activity differences in PKCα between the mice aged 2, 12, or 24 mo. However, we demonstrated a significantly higher PKCε activity in the mice at 12 and 24 mo than the in the mice 2 mo of age. The increase in activity is likely due to a nearly threefold increase in PKCε protein in the lung during aging. To strengthen the connection between activation of PKCε and ciliary slowing, we treated tracheas of mice at 2 mo with the PKCε agonist 8-[2-(2-pentylcyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA). We noted a similar decrease in baseline CBF, and the cilia remained sensitive to stimulation with ß2 agonists. The mechanisms for the slowing of baseline CBF have not been previously determined. In this mouse model of aging we were able to show that decreases in CBF are related to an increase in PKCε activity.

Decreased home cage movement and oromotor impairments in adult Fmr1-KO mice.

Fragile X syndrome (FXS) is a common inherited disorder that significantly impacts family and patient day-to-day living across the entire life span. The childhood and adolescent behavioral consequences of FXS are well appreciated. However, there are significantly fewer studies (except those examining psychiatric comorbidities) assessing behavioral phenotypes seen in adults with FXS. Mice engineered with a genetic lesion of fragile X mental retardation 1 (Fmr1) recapitulate important molecular and neuroanatomical characteristics of FXS, and provide a means to evaluate adult behavioral phenotypes associated with FXS. We give the first description of baseline behaviors including feeding, drinking, movement and their circadian rhythms; all observed over 16 consecutive days following extensive environmental habituation in adult Fmr1-KO mutant mice. We find no genotypic changes in mouse food ingestion, feeding patterns, metabolism or circadian patterns of movement, feeding and drinking. After habituation, Fmr1-KO mice show significantly less daily movement during their active phase (the dark cycle). However, Fmr1-KO mice have more bouts of activity during the light cycle compared with wild types. In addition, Fmr1-KO mice show significantly less daily water ingestion during the circadian dark cycle, and this reduction in water intake is accompanied by a decrease in the amount of water ingested per lick. The observed water ingestion and circadian phenotypes noted in Fmr1-KO mice recapitulate known clinical aspects previously described in FXS. The finding of decreased movement in Fmr1-KO mice is novel, and suggests a dissociation between baseline and novelty-evoked activity for Fmr1-KO mice.

Mouse models of serotonin receptor function: toward a genetic dissection of serotonin systems.

Central serotonin (5-hydroxytryptamine, 5-HT) systems regulate a wide variety of complex behaviors, and are targeted by drugs used in the treatment of diverse neuropsychiatric disorders. The actions of 5-HT are mediated by a large and heterogeneous family of 5-HT receptor subtypes. Studies of the functional significance of individual subtypes have been complicated by the limited availability of selective receptor agonist and antagonist drugs. Molecular genetic techniques offer complementary approaches for studying the behavioral roles of individual 5-HT receptor subtypes through the generation of gene-targeted and transgenic lines of mice with altered expression of 5-HT receptor genes. This review will examine insights into the serotonergic regulation of behavior that have been produced by the study of these lines, as well as discuss important caveats to the interpretation of these studies.

Validation of Flow Cytometry and Magnetic Bead-Based Methods to Enrich CNS Single Cell Suspensions for Quiescent Microglia.

Microglia are resident mononuclear phagocytes within the CNS parenchyma that intimately interact with neurons and astrocytes to remodel synapses and extracellular matrix. We briefly review studies elucidating the molecular pathways that underlie microglial surveillance, activation, chemotaxis, and phagocytosis; we additionally place these studies in a clinical context. We describe and validate an inexpensive and simple approach to obtain enriched single cell suspensions of quiescent parenchymal and perivascular microglia from the mouse cerebellum and hypothalamus. Following preparation of regional CNS single cell suspensions, we remove myelin debris, and then perform two serial enrichment steps for cells expressing surface CD11b. Myelin depletion and CD11b enrichment are both accomplished using antigen-specific magnetic beads in an automated cell separation system. Flow cytometry of the resultant suspensions shows a significant enrichment for CD11b(+)/CD45(+) cells (perivascular microglia) and CD11b(+)/CD45(-) cells (parenchymal microglia) compared to starting suspensions. Of note, cells from these enriched suspensions minimally express Aif1 (aka Iba1), suggesting that the enrichment process does not evoke significant microglial activation. However, these cells readily respond to a functional challenge (LPS) with significant changes in the expression of molecules specifically associated with microglia. We conclude that methods employing a combination of magnetic-bead based sorting and flow cytometry produce suspensions highly enriched for microglia that are appropriate for a variety of molecular and cellular assays.

COMT Val158Met genotype influences neurodegeneration within dopamine-innervated brain structures.

OBJECTIVE: We sought to determine whether the Val(158)Met polymorphism in the catechol-O-methyltransferase (COMT) gene influences neurodegeneration within dopamine-innervated brain regions. METHODS: A total of 252 subjects, including healthy controls and patients with Alzheimer disease, behavioral variant frontotemporal dementia, and semantic dementia, underwent COMT genotyping and structural MRI. RESULTS: Whole-brain voxel-wise regression analyses revealed that COMT Val(158)Met Val allele dosage, known to produce a dose-dependent decrease in synaptic dopamine (DA) availability, correlated with decreased gray matter in the region of the ventral tegmental area (VTA), ventromedial prefrontal cortex, bilateral dorsal midinsula, left dorsolateral prefrontal cortex, and right ventral striatum. Unexpectedly, patients carrying a Met allele showed greater VTA volumes than age-matched controls. Gray matter intensities within COMT-related brain regions correlated with cognitive and behavioral deficits. CONCLUSIONS: The results are consistent with the hypothesis that increased synaptic DA catabolism promotes neurodegeneration within DA-innervated brain regions.

Mechanical actions of heterogenic reflexes linking long toe flexors with ankle and knee extensors of the cat hindlimb.

1. To study the means whereby ankle biomechanics are represented in the interneuronal circuitry of the spinal cord we examined stretch-evoked reflex interactions between the physiological extensors flexor hallucis longus (FHL) and flexor digitorum longus (FDL) as well as their interactions with gastrocnemius (G), soleus (S), and the quadriceps group (Q) in 34 unanesthetized decerebrate cats. To evoke stretch, DC motors provided ramp-hold-release length changes to tendons detached from their bony insertions. Semiconductor myographs measured resultant muscle force response. Reflexes were examined under both quiescent (no active force generation) and activated conditions; muscle activation was achieved through either crossed-extension or flexion reflexes. 2. FHL and FDL share mutual excitatory stretch-evoked interactions under most conditions examined. These interactions depended on muscle length, were asymmetric (with FHL contributing a larger magnitude of reflex excitation onto FDL), and occurred at a latency of 16 ms. Mutual Ia synergism previously described for these two muscles provides a basis for all of the above findings. Our data demonstrate that for this muscle pair, reflex connectivities revealed at the intracellular level can be extrapolated to cover the entire motoneuron pool; further, our data directly demonstrate the net mechanical result of ensemble synaptic events. 3. FHL was found to share strong, mutually inhibitory stretch-evoked interactions with G, S, and Q. Stepwise regression statistical analyses determined that these interactions depended on recipient muscle force and donor muscle force. These reflex interactions all occurred at a latency of 28 +/- 4 (SE) ms. Further, the heterogenic inhibition between FHL/G and FHL/S was attenuated by strychnine infusion (intravenous) but unaffected by either mecamylamine, picrotoxin, or baclofen infusion (intravenous, intrathecal). Disynaptic Ib inhibition previously described among hindlimb extensors provides a basis for the above findings; our data demonstrate that under certain conditions the ensemble activity of this system can cause a dramatic decline in whole muscle force output. 4. By contrast, FDL was found to share mutually inhibitory, stretch-evoked reflex interactions with G, S, and Q that were much weaker than those observed between FHL and these same muscles. The small magnitude of inhibition observed in these interactions made it difficult to assess reflex latency or to determine the factor(s) that best predicted the heterogenic inhibition. 5. This study provides further evidence of intrinsic differences in interneuronal organization between muscles whose activity occurs in a periodic manner during locomotion ("stereotypical") and a muscle whose locomotor activity is characterized by both periodic and nonperiodic components ("facultative").(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanical actions of heterogenic reflexes among ankle stabilizers and their interactions with plantarflexors of the cat hindlimb.

1. The stretch-evoked reflex organization of muscles whose major action is to abduct [peroneus brevis (PB); peroneus longus (PL)] and adduct [tibialis posterior (TP); flexor digitorum longus (FDL); flexor hallucis longus (FHL)] the ankle, and their interactions with the hindlimb extensors gastrocnemius (G) and soleus (S), were studied in 27 unanesthetized decerebrate cats. Ramp-hold-release stretches of physiological amplitudes were applied to muscle tendons detached from their bony insertion, and muscle force output was measured in response to these perturbations. Flexion and crossed-extension reflexes were used to modulate baseline force. 2. PB and TP shared strong, length-dependent, short-latency inhibitory reflexes prominent when the muscles were either actively generating force or quiescent. The mechanical characteristics of this reflex suggest Ia reciprocal inhibition as the underlying mechanism. Just as reciprocal inhibition between S and tibialis anterior stiffens the ankle joint against sagittal perturbations, we propose that reciprocal inhibition between PB and TP stiffens the ankle joint against nonsagittal perturbations. 3. In all preparations (n = 7) and under all conditions examined, PB and PL shared well-demonstrated mutual excitation. The reflex responses were asymmetric (favoring excitation of PL), length dependent, and occurred simultaneously with the stretch reflex at a latency of 16-18 ms. Mutual monosynaptic projections previously described between these two muscles explain all of the above findings. Our data further demonstrate that, under certain conditions, the ensemble activity of this reflex interaction has a powerful effect on the mechanical behavior of the muscle. 4. The heterogenic reflex organization of the ankle adductors was as follows: FDL evoked a modest excitation on TP, whereas FHL evoked weak inhibition. Latency of the excitation from FDL onto TP (24 ms) was greater than expected if the reflex were mediated by heteronymous Ia afferents. In all preparations examined (n = 3), TP contributed no significant reflexes onto either FDL or FHL. 5. Mutual, asymmetric inhibition characterized interactions between PB and the plantarflexors S and G. Most remarkable was a novel, long-latency (72-74 ms) reflex inhibition evoked on both S and G by stretch of PB. When this inhibition occurred, it dramatically decreased the S (or G) stretch response. Longer PB lengths evoked greater inhibition of isometric S; regression analysis indicated that the model best predicting this inhibition contained muscle force and stiffness terms. No long-latency reflexes were noted from either G or S onto PB. The mechanism underlying long-latency inhibition is presently unknown; however, features of this interaction suggest interneurons receive either group II or group III afferent input. 6. G and TP shared short latency, mutually inhibitory, asymmetric reflexes favoring inhibition of TP. No long-latency interactions were noted, nor were there any mechanically significant interactions between S and TP. 7. Reflex interactions across the abduction/adduction axis thus favored inhibition of plantarflexion and adduction torques while emphasizing abduction torques: PB/S (or PB/G) interactions were mutual, asymmetric, and favored inhibition of G and S; TP/G interactions were mutual, asymmetric, and favored inhibition of TP; TP/PB interactions were approximately balanced. The overall mechanical outcome of these inhibitory interactions may partly underlie the global corrective strategy seen in intact cats subjected to linear perturbations. 8. No significant reflex interactions were demonstrated between PL and TP, G, or S, nor were any long-latency reflexes noted. Thus, whereas reflex interactions between the stereotypically activated PB and other stereotypically activated muscles (including TP, G, and S) were strong and well-demonstrated, interactions between the variably activated PL and these same muscles were far weaker.

Control of torque direction by spinal pathways at the cat ankle joint.

To study the biomechanics of the calcaneal tendon's complex insertion onto the calcaneus, we measured torque-time trajectories exerted by the triceps surae and tibialis anterior muscles in eight unanesthetized decerebrate cats using a multi-axis force-moment sensor placed at the ankle joint. The ankle was constrained to an angle of 110 degrees plantarflexion. Muscles were activated using crossed-extension (XER), flexion (FWR), and caudal cutaneous sural nerve (SNR) reflexes. Torque contributions of other muscles activated by these reflexes were eliminated by denervation or tenotomy. In two animals, miniature pressure transducers were implanted among tendon fibers from the lateral gastrocnemius (LG) muscle that insert straight into the calcaneus or among tendon fibers from the medial gastrocnemius (MG) that cross over and insert on the lateral aspect of calcaneus. Reflexively evoked torques had the following directions: FWR, dorsiflexion and adduction; SNR, plantarflexion and abduction; and XER, plantarflexion and modest abduction or adduction. The proportion of abduction torque to plantarflexion torque was always greater for SNR than XER; this difference was about 50% of the magnitude of abduction torque generated by tetanic stimulation of the peronei. During SNR, pressures were higher in regions of the calcaneal tendon originating from MG than regions originating from LG. Similarly, pressures within the MG portion of the calcaneal tendon were higher during SNR than during XER, although these two reflexes produced matched ankle plantarflexion forces. Selective tenotomies and electromyographic recordings further demonstrated that MG generated most of the torque in response to SNR, while soleus, LG, and MG all generated torques in response to XER.(ABSTRACT TRUNCATED AT 250 WORDS)

Reinnervated muscles fail to produce stretch reflexes.

1. We studied the stretch-evoked reflex organization of hind limb muscles in two decerebrate cats 36 mo after unilateral section and immediate surgical repair of the common nerve supplying the lateral gastrocnemius (LG) and soleus (S) muscles. 2. The production of considerable reflex force by reinnervated muscles in response to electrical stimulation of uninjured nerves indicated substantial functional recovery of motor units. However, reduction in the responsiveness of reinnervated muscles to stretch of the untreated medial gastrocnemius (MG) muscle indicated some deficit in recovery of normal synaptic integration. 3. Stretch failed to elicit autogenic excitation of the reinnervated S and LG. This failure was observed whether the reinnervated muscles were quiescent or contracting in other reflexes. 4. The heterogenic reflex organization of reinnervated muscles was abnormal. Stretch of the reinnervated S failed to evoke heterogenic reflexes both in the untreated MG and in the reinnervated LG. Stretch of the reinnervated LG failed to produce excitation of MG. 5. These findings demonstrate deficiencies in proprioceptive feedback from reinnervated muscles and lead us to expect incomplete recovery of motor function after nerve section.