Susan E. Smittkamp

Selective changes in nocifensive behavior despite normal cutaneous axon innervation in leptin receptor–null mutant (db/db) mice

Abstract  Much of our understanding of the effects of diabetes on the peripheral nervous system is derived from models induced by streptozotocin in which hyperglycemia is rapidly caused by pancreatic beta‐cell destruction. Here, we have quantified sensory impairments over time in leptin receptor (lepr)–null mutant (−/−) mice, a type 2 model of diabetes in which the absence of leptin receptor signaling leads to obesity and chronic hyperglycemia by 4 weeks of age. To assess these mice as a model for peripheral neuropathy, we quantified the responsiveness of lepr (−/−) mice to mechanical, thermal, and chemogenic stimuli, as well as epidermal and dermal innervation of the hind paw. Compared with wild‐type (+/+) and heterozygous (+/−) mice, lepr (−/−) mice displayed reduced sensitivity to mechanical stimuli by 6 weeks of age, and however, responses to noxious heat were normal. Lepr (−/−) mice also devoted less activity to their injected paw during the second phase following formalin administration. However, epidermal and dermal innervation of lepr (−/−) mice was not different from that of lepr (+/+) and (+/−) mice even after 10 weeks of hyperglycemia, suggesting that cutaneous innervation is resistant to chronic hyperglycemia in these mice. These results suggest that certain rodent nocifensive behaviors may be linked to the abundance of cutaneous innervation, while others are not. Finally, these results reveal that the lepr (−/−) mice may not be useful to study neuropathy associated with distal axonal degeneration but may be better suited for studies of hyperglycemia‐induced sensory neuron dysfunction without distal nerve loss.

TIME-COURSE AND CHARACTERIZATION OF OROLINGUAL MOTOR DEFICITS IN B6SJL-Tg(SOD1-G93A)1GUR/J MICE

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease affecting upper and lower motor neurons. Symptom onset may occur in the muscles of the limbs (spinal onset) or those of the head and neck (bulbar onset). Bulbar involvement is particularly important in ALS as it is associated with increased morbidity and mortality. The purpose of this study was to characterize bulbar motor deficits in the B6SJL-Tg(SOD1-G93A)1Gur/J (SOD1-G93A) mouse model of familial ALS. We measured orolingual motor function by placing thirsty mice in a customized operant chamber that allows for measurement of tongue force and lick rhythm as animals lick water from an isometric disc. Testing spanned the pre-symptomatic, symptomatic, and end-stage segments of the disease. Rotarod performance, fore- and hindlimb grip strength, and locomotor activity were also monitored regularly during this period. We found that spinal involvement was apparent first, with both fore- and hindlimb grip strength being affected in SOD1-G93A mice from the onset of testing (64 days of age). Rotarod performance was affected by 71 days of age. Locomotor activity was not affected, even near end-stage. Bulbar involvement appeared much later, with tongue motility being affected by 100 days of age. Tongue force was affected by 115 days of age. To our knowledge, these findings are the first to describe the onset of bulbar versus spinal motor signs and characterize orolingual motor deficits in this preclinical model of ALS.

Measures of bulbar and spinal motor function, muscle innervation, and mitochondrial function in ALS rats

Symptom onset in amyotrophic lateral sclerosis (ALS) may occur in the muscles of the limbs (spinal onset) or those of the head and neck (bulbar onset). Most preclinical studies have focused on spinal symptoms, despite the prevalence of and increased morbidity and mortality associated with bulbar disease. We measured lick rhythm and tongue force to evaluate bulbar disease in the SOD1-G93A rat model of familial ALS. Body weight and grip strength were measured concomitantly. Testing spanned the early (maturation), middle (pre-symptomatic), and late (symptomatic and end-stage) phases of the disease. We measured a persistent tongue motility deficit that became apparent in the early phase of the disease, providing behavioral evidence of bulbar pathology. At end-stage, however, cytochrome oxidase (CO) activity was normal in the hypoglossal nucleus, and in the tongue, neuromuscular innervation, citrate synthase (CS) protein levels and activity, and uncoupling protein 3 (UCP3) protein levels remained unchanged. Interestingly, significant denervation and atrophy were evident in the end-stage sternomastoid muscle, providing peripheral anatomical evidence of bulbar pathology. Changes in body weight and grip strength occurred in the late phase of the disease. Extensive atrophy and denervation were observed in the end-stage gastrocnemius muscle. In contrast to our findings in the tongue, CS protein levels were decreased in the extensor digitorum longus (EDL) and soleus, although CS activity was maintained or increased. UCP3 protein was decreased also in the EDL. These data provide evidence of differential effects in muscles that were more or less affected by disease.

Age-related changes in orolingual motor function in F344 vs F344/BN rats.

Normal aging is associated with both locomotor and orolingual motor deficits. Preclinical studies of motor function in normal aging, however, have focused primarily on locomotor activity. The purpose of this study was to measure age-related changes in orolingual motor function and compare these changes between two rat strains commonly used in aging studies: Fischer 344 (F344) and Fischer 344/Brown Norway hybrid (F344/BN) rats. Rats (6-, 12-, 18- and 24-months of age) were trained to lick water from an isometric force-sensing operandum so that the number of licks per session, licking rhythm (licks/second) and lick force could be measured. In both strains, the number of licks per session was greatest in the oldest group, while this measure was greater for F344/BN rats at all ages. Peak tongue force increased with age in F344/BN rats, did not change with age in the F344 rats, and was greater for the F344/BN rats at all ages. Both strains exhibited an age-related slowing of licking rhythm beginning with the 18-month-old group. These findings suggest that despite lifespan differences between these two rat strains, diminished tongue motility emerges at the same age.

Time course and quantification of changes in cochlear integrity observed in commercially raised broiler chickens.

Extensive cochlear hair cell damage and loss occurs in aged broiler chickens. We describe the time course and several characteristics of this decline in cochlear integrity in 19-, 30-, 38- and 66-week-old commercially raised broiler chickens. The 19-week-old group is normal and serves as a baseline for comparison. Generally, cochlear damage increases in severity and percent length of the cochlea with age. Hair cell density increases from the base to the apex. Density is similar across the groups in regions of the cochlea that sustained little or no damage, and decreases in regions of extreme damage. Counts of normal and abnormal hair cells are inversely related. A subset of 66-week-old birds has higher density measurements and increased hair cell counts in the apical region of the cochlea. The progressive damage found in these commercially raised birds is described in the context of both the effects of age and noise exposure on the auditory system. Two additional groups of birds were raised at the University of Kansas Medical Center in a quieter environment to determine the cause of the damage seen in age-matched commercially raised birds. These cochleae are largely normal; a small number displayed damage. This suggests that noise exposure exacerbates naturally occurring cochlear degradation.

SOD1-G93A mice exhibit muscle-fiber-type-specific decreases in glucose uptake in the absence of whole-body changes in metabolism.

Background: Skeletal muscles play an important role in systemic glucose homeostasis and are purported to be the origin of the altered metabolic state observed in amyotrophic lateral sclerosis (ALS). Objective: The purpose of this study was to evaluate whole-body and muscle-specific glucose metabolism in the SOD1-G93A mouse model of ALS. Methods: We assessed glucose tolerance in early-, middle-, and late-stage SOD1-G93A and control mice using an intraperitoneal glucose tolerance test. We then measured the respiratory exchange ratio (CO2 production/O2 consumption) as a function of fasting and feeding using indirect calorimetry in a subset of male mice at these time points. Finally, muscles from all mice were harvested to evaluate basal and insulin-stimulated glucose transport in fast- and slow-twitch muscles. Results: No changes in systemic glucose clearance were observed in SOD1-G93A mice at any stage, nor were there changes in fasting insulin levels. Indirect calorimetry revealed an increase in the respiratory exchange ratio during the fed state at middle, but not at early or late stages of disease. Middle-stage SOD1-G93A mice exhibited decreased insulin-stimulated glucose uptake in fast-twitch, but not slow-twitch, skeletal muscle. Late-stage SOD1-G93A mice exhibited decreased insulin-stimulated glucose uptake in both fast- and slow-twitch muscle, as well as increased basal (non-insulin-stimulated) glucose uptake. Conclusions: These results suggest that alterations in muscle metabolism occur in a fiber-type-specific manner in ALS, but do not necessarily lead to whole-body metabolic changes in SOD1-G93A mice.

Effects of age and cochlear damage on the metabolic activity of the avian cochlear nucleus.

Most aging commercially raised broiler chickens display a progressive loss of cochlear hair cells in a pattern similar to the cochlear degeneration found in aging humans: basal (high frequency) hair cells are affected first, followed by apical (low frequency) hair cells [Durham et al., Hear. Res. 166 (2002) 82-95]. Here, cochlear anatomy was assessed from scanning electron micrographs. Then, the metabolic activity of cochlear nucleus (nucleus magnocellularis, NM) neurons in 15-19, 30, 39, 40, and 65-66 week old broiler chickens was examined using cytochrome oxidase histochemistry and compared to the degree of cochlear abnormality. Cochleae of 15-19 week old birds are largely normal; therefore the level of NM metabolic activity is considered the baseline. Cochleae of the 30 week old group display mild damage and hair cell regeneration in the base. Metabolic activity in rostral (high frequency) NM is increased relative to the baseline, while activity remains unchanged in caudal (low frequency) NM. The 39 and 65-66 week old groups display severe and total damage extending into the apex of the cochlea. Metabolic activity is decreased in rostral and caudal NM at these ages. These results suggest that auditory central nervous system metabolism (cytochrome oxidase activity) is affected by changes in the aging chicken cochlea.

Predicting severity of cochlear hair cell damage in adult chickens using DPOAE input–output functions

Distortion product otoacoustic emissions (DPOAE) were recorded from the ear canal of aged broiler chickens which have been shown to present with age-related cochlear degeneration [Hear. Res. 166 (2002) 82]. We describe the relationship between the shape of the DPOAE input-output (I/O) function and the type of hair cell damage present at and between the cochlear frequency places of the DPOAE primary tones (f1 and f2). The mid stimulus level compressive growth of the mean DPOAE I/O functions is reduced in a graded fashion relative to the severity of hair cell damage. However, individual DPOAE I/O functions within most hair cell damage groups show large variability from this characteristic. Various least squares regression models were used to predict hair cell density from indices derived from the DPOAE I/O function (area, threshold and slope). The results showed that no simple linear relationship exists between hair cell density and the DPOAE I/O function indices. Multivariate binary logistic regression used DPOAE I/O function indices to predict membership in hair cell damage groups. The logistic model revealed that DPOAE threshold can be used to predict the occurrence of severe/total hair cell damage with good specificity though poor sensitivity.

Role of cochlear integrity in cochlear nucleus glucose metabolism and neuron number after cochlea removal in aging broiler chickens

In the chicken auditory system, cochlear nucleus (nucleus magnocellularis, NM) neurons receive their only excitatory input from the ipsilateral cochlea. Cochlea removal (CR) results in an immediate decrease in NM neuron electrical activity, followed by death of approximately 30% of NM neurons. Previous work showed a decrease in NM activity and subsequent loss of NM neurons in all chicks. Egg layer adults showed NM neuron loss after CR, while neuron number remained stable in broiler adults. This suggested that effects of CR on NM were age- and breed-dependent. We now know that most aging egg layer chickens maintain largely normal cochleae throughout adulthood. Some exhibit cochlear damage with age. The converse is true of broiler chickens. Most aging broiler chickens display cochlear degeneration, with some maintaining normal cochlear anatomy throughout adulthood. The presence of extensive cochlear damage may alter the effect of CR on NM, leading to the described differences. Here, we examine the effect of unilateral CR on NM glucose metabolism and neuron number in 2, 30, 39, and 52 week-old broiler chickens found to have normal cochleae. Chickens with damaged cochleae were excluded. Using 2-deoxyglucose uptake to evaluate bilateral NM glucose metabolism, we found significantly decreased uptake ipsilateral to CR at each age examined. Bilateral cell counts revealed significant neuron loss ipsilateral to CR at each age examined. This suggests that NM glucose metabolism decreases and subsequent neuron death occurs in aging broiler chickens when a normal cochlea is removed. The status of the cochlea must play a role in the effect of deafferentation on NM glucose metabolism and neuron survival. The effect of CR appears to be dependent upon neither age nor breed, but upon cochlear integrity instead.

Contributions of age, cochlear integrity, and auditory environment to avian cochlear nucleus metabolism

Most commercially raised broiler chickens display progressive cochlear degeneration with age [Hear. Res. 166 (2002) 82]. Recent work examining the effects of age and cochlear degeneration on avian cochlear nucleus (nucleus magnocellularis, NM) metabolism showed that changes in metabolic activity occur with age and cochlear damage [Hear. Res. 175 (2003) 101]. The auditory environment also differed between facilities housing young and adult birds. The relative contributions of age, cochlear degeneration, and auditory environment to these changes in NM metabolism are unknown. Using cytochrome oxidase (CO) histochemistry, NM neuron metabolism is examined in several age groups of birds under varying conditions. When normal cochlear integrity and auditory environment are held constant, CO staining is significantly decreased in adult vs. young birds. When age and auditory environment are held constant, CO staining is significantly decreased in birds with damaged vs. normal cochleae. When age and normal cochlear integrity are held constant, CO staining is significantly decreased in birds living in a quiet vs. noisy environment. All factors examined cause changes in CO staining, which is indicative of NM metabolic activity. Results are discussed in the context of mitochondrial aging, afferent regulation, and auditory deprivation and enrichment.