The laminar organization of cell types in macaque cortex and its relationship to neuronal oscillations.

The canonical microcircuit (CMC) has been hypothesized to be the fundamental unit of information processing in cortex. Each CMC unit is thought to be an interconnected column of neurons with specific connections between excitatory and inhibitory neurons across layers. Recently, we identified a conserved spectrolaminar motif of oscillatory activity across the primate cortex that may be the physiological consequence of the CMC. The spectrolaminar motif consists of local field potential (LFP) gamma-band power (40-150 Hz) peaking in superficial layers 2 and 3 and alpha/beta-band power (8-30 Hz) peaking in deep layers 5 and 6. Here, we investigate whether specific conserved cell types may produce the spectrolaminar motif. We collected laminar histological and electrophysiological data in 11 distinct cortical areas spanning the visual hierarchy: V1, V2, V3, V4, TEO, MT, MST, LIP, 8A/FEF, PMD, and LPFC (area 46), and anatomical data in DP and 7A. We stained representative slices for the three main inhibitory subtypes, Parvalbumin (PV), Calbindin (CB), and Calretinin (CR) positive neurons, as well as pyramidal cells marked with Neurogranin (NRGN). We found a conserved laminar structure of PV, CB, CR, and pyramidal cells. We also found a consistent relationship between the laminar distribution of inhibitory subtypes with power in the local field potential. PV interneuron density positively correlated with gamma (40-150 Hz) power. CR and CB density negatively correlated with alpha (8-12 Hz) and beta (13-30 Hz) oscillations. The conserved, layer-specific pattern of inhibition and excitation across layers is therefore likely the anatomical substrate of the spectrolaminar motif. Significance Statement: Neuronal oscillations emerge as an interplay between excitatory and inhibitory neurons and underlie cognitive functions and conscious states. These oscillations have distinct expression patterns across cortical layers. Does cellular anatomy enable these oscillations to emerge in specific cortical layers? We present a comprehensive analysis of the laminar distribution of the three main inhibitory cell types in primate cortex (Parvalbumin, Calbindin, and Calretinin positive) and excitatory pyramidal cells. We found a canonical relationship between the laminar anatomy and electrophysiology in 11 distinct primate areas spanning from primary visual to prefrontal cortex. The laminar anatomy explained the expression patterns of neuronal oscillations in different frequencies. Our work provides insight into the cortex-wide cellular mechanisms that generate neuronal oscillations in primates.

Adaptation of nicotinic acetylcholine receptor, myogenin, and MRF4 gene expression to long-term muscle denervation.

Muscle activity alters the expression of functionally distinct nicotinic acetylcholine receptors (nAChR) via regulation of subunit gene expression. Denervation increases the expression of all subunit genes and promotes the expression of embryonic-type (alpha 2 beta delta gamma) nAChRs, while electrical stimulation of denervated muscle prevents this induction. We have discovered that the denervation-induced increases in alpha, beta, gamma, and delta subunit gene expression do not persist in muscles that have been denervated for periods extending beyond a couple of months. However, expression of RNA encoding the epsilon-subunit remains elevated suggesting a return to expression of predominantly adult-type (alpha 2 beta delta epsilon) nAChR in long-term denervated muscles; a finding confirmed by single channel patch-clamp analysis. Since the nAChR subunit genes are regulated by the MyoD family of muscle regulatory factors, and the genes encoding these factors are also induced following short-term muscle denervation, we determined their level of expression in long-term denervated muscle. Although MyoD and myf-5 RNA levels remained elevated, myogenin and MRF4 RNAs were induced only transiently by muscle denervation. Surprisingly, Id-1, a negative regulator of transcription, was gradually induced in denervated muscle with RNA levels peaking about two months after denervation. It is likely that this maintained level of increased Id expression, in conjunction with the returning levels of myogenin and MRF4 expression, account for the reduced level of embryonic receptors in long-term denervated muscle. These changing patterns of gene expression may have important consequences for the ability of muscle to recover function after denervation.

Induction of adult-type nicotinic acetylcholine receptor gene expression in noninnervated regenerating muscle.

Expression of adult-type nicotinic acetylcholine receptors at the neuromuscular junction is thought to result from selective induction of their genes in endplate-associated nuclei due to local neurotrophic control. However, denervation studies indicate that endplate-specific expression can be maintained in the absence of the nerve. We investigated the role played by the basal lamina in this expression by assaying for the adult-type-specific epsilon RNA in noninnervated regenerating muscle. We found that this RNA is locally expressed beneath the old endplates after 10 days of regeneration. At earlier times epsilon RNA is also found in areas other than the endplate region. These results indicate that in adult muscle the basal lamina contains all the components necessary to direct nicotinic acetylcholine receptor gene expression to the endplate.

Muscle fiber branching--difference between grafts in old and young rats.

Large numbers of branched muscle fibers occur in the freely grafted rat extensor digitorum longus muscle. The ratio of branched/non-branched muscle fibers in grafts is much higher in old (24 months) than in young (4 months) host rats. Cross-age transplants show that the proportion of branched muscle fibers is related to the age of the grafted muscle and not to the age of the host. This is in contrast to mass and maximum isometric tension, in which the age of the host, rather than the age of the grafted muscle, is the determinant of the success of the muscle graft.

Alkaline phosphatase and dipeptidylpeptidase IV staining of tissue components of skeletal muscle: a comparative study.

A combined alkaline phosphatase (AP) and dipeptidlypeptidase IV (DPP IV) staining reaction has demonstrated enzymatic heterogeneity of the arterial and venous segments of capillaries in rat skeletal muscle. This study compared the staining reactions of skeletal muscles in many commonly used laboratory animals, including the axolotl, chick, quail, Monodelphys, rat, mouse, hamster, guinea pig, rabbit, dog, monkey, and human. DPP IV activity was found in the venous ends of the capillaries and in the endothelium of some larger veins in many of the species but was never demonstrated in the arterial side of the circulation. AP was found in the arterial ends of capillaries in all species except the axolotl, and it was also found in the endothelium of larger arteries of most species. AP activity was absent in venous endothelium of all species except for birds and Monodelphys. DPP IV activity was found in the perineurium of intramuscular nerves of most species, and AP activity was commonly seen in tendons and intramuscular connective tissue. The interspecies variability found in this study shows that care must be taken in comparing experimental data involving this technique from one species to another, but within a species the technique allows a fine level of discrimination between functionally distinct compounds of skeletal muscle tissue.

Ultrastructure of mepivacaine-induced damage and regeneration in rat extraocular muscle.

Adult rats were given a single retrobulbar injection of 50 microliters of 2.0% mepivacaine and the lateral rectus muscles were examined ultrastructurally at intervals from 15 min to 30 days post-injection. There were three purposes of the study: (1) to determine the extent of muscle fiber damage caused by the anesthetic; (2) to document the subsequent course of muscle fiber regeneration; and (3) to relate these findings to clinical data on possible adverse effects of local anesthetics on human extraocular muscle function. The lateral rectus muscle was massively damaged by exposure to the anesthetic, with membrane lesions seen as early as 15 min after the injection. Intracellular damage was followed by the phagocytic removal of the remnants of the damaged muscle fibers. The activation of satellite cells to myoblasts began during the phase of phagocytosis, and between 3 and 4 days after injection multinucleated myotubes actively forming sarcomeres appeared. Even during later stages of muscle fiber regeneration, evidence of damage was seen in muscle fibers that were not destroyed during the first 2 days post-injection. The results of this experiment show (1) that the vast majority of lateral rectus muscle fibers are rapidly broken down by the anesthetic, but that the destroyed muscle fibers are replaced by regenerating ones; and (2) that the ultrastructure of regeneration of extraocular muscle fibers differs little from the regeneration of somatic muscle fibers. The myotoxic effects of retrobulbarly applied local anesthetics in rats seem to be much greater than they are in primates.

Postoperative diplopia and ptosis. A clinical hypothesis based on the myotoxicity of local anesthetics.

Postoperative diplopia and ptosis can be temporary or permanent complications in patients who have undergone ophthalmic surgery while under local anesthesia. We encountered six patients with such complications and hypothesize that some cases of postoperative diplopia and ptosis could be attributed to myotoxic effects of local anesthetics. These effects may cause the degeneration and subsequent regeneration of muscle fibers of the levator or extraocular muscles and result in temporary or permanent muscle weakness.

Rat extraocular muscle regeneration. Repair of local anesthetic-induced damage.

Local anesthetics that are commonly used in ophthalmic surgery (0.75% bupivacaine hydrochloride, 2.0% mepivacaine hydrochloride, and 2.0% lidocaine hydrochloride plus 1:100,000 epinephrine) were injected into the retrobulbar area of rat eyes. Controls were injected with physiological saline. All three anesthetics produced massive degeneration of the extraocular muscles. Muscle degeneration is followed by regeneration of the damaged muscle fibers. In addition to muscle damage, severe damage was also seen in harderian glands, especially after exposure to mepivacaine and lidocaine plus epinephrine. With these findings in rats, it is hypothesized that the temporary diplopia sometimes seen in patients after ophthalmic surgery might be due to anesthetic-induced damage to the extraocular muscles.

Studies on the regenerative recovery of long-term denervated muscle in rats.

Denervated extensor digitorum longus (EDL) muscles in rats rapidly lose mass and contractile force. After two months of denervation, mass and maximum tetanic force have fallen to 31% and 2% of the values of contralateral control muscles. Our purpose was to determine if grafting a long-term denervated muscle into an innervated site provides an effective means of restoring its structure and function. EDL muscles that had been denervated for periods of 2-12 months were freely grafted into innervated sites of EDL muscles in 4-month inbred host animals. Contralateral normally innervated EDL muscles from the same donors were implanted into the opposite legs of the same hosts. Two months after grafting, the muscles were removed and measurements were made in vitro of isometric contractile properties. The grafts were then prepared for morphological analysts. In all cases, the maximum forces generated by innervated grafts of denervated muscles were greater than those generated by denervated muscles. However, when compared with grafts of control muscles in the contralateral limb, grafts of previously denervated muscles showed a steady decline in structural and functional recovery corresponding to the time of previous denervation. The decline was especially pronounced for muscles denervated between 2 and 7 months prior to grafting. Grafts of 7-month denervated muscles were restored to only 17% of the maximum tetanic force of contralateral control grafts compared with 83% for grafts of 2-month denervated muscles. The longer a muscle had been denervated prior to grafting, the higher proportion of thin atrophic muscle fibers it contained. We conclude that grafting into an innervated site improves the mass and maximum force of a muscle over the denervated state, but the longer the period of prior denervation the poorer the recovery of the grafted muscles.

Factors influencing the repair and adaptation of muscles in aged individuals: satellite cells and innervation.

From the standpoint of structure, the loss of muscle mass could be attributed to the loss of muscle fibers, the reduction of volume of persisting muscle fibers, or both. This review will concentrate upon factors that could contribute to the presence of thin muscle fibers in old muscles. One mechanism that could account for the presence of a population of thin muscle fibers is the loss of innervation due to the death or the remodeling of motor units in aging muscles. If the denervated muscle fibers fail to become reinnervated, or if they are unable to respond to reinnervation by new nerve terminals, they would undergo a progressive atrophy. If muscle fibers are damaged through exercise, direct trauma or other causes, they would degenerate and regenerate. Early regenerating muscle fibers are thinner than normal. If the regenerating muscle fibers are non-innervated, regeneration will not be complete; instead the regenerating muscle fibers will atrophy. All of the above mechanisms could contribute to an overall reduction in muscle mass. This review ends with an enumeration of outstanding questions concerning muscle atrophy and its reversal in old individuals.

The regeneration of noninnervated muscle grafts and marcaine-treated muscles in young and old rats.

Free grafts of the extensor digitorum longus (EDL) muscle in 4-month-old rats regenerate 2-3 times better than in 24-month-old rats. Based on these data, we formulated the working hypothesis that deficient reinnervation is one of the most important age-related environmental factors within the host that might account for the poor regeneration. In the present experiments, we compared the regeneration of EDL muscles in two groups of young and old rats: (a) 21-day grafts, with fibers regenerating in the absence of nerves, and (b) Marcaine-treated muscle with fibers regenerating in the presence of uninterrupted innervation. The specific hypothesis was that, under each of these circumstances, reinnervation was not involved and age-related differences in regeneration would not be seen. Differences were assessed by measurements of mass and maximum isometric force normalized to values for age-matched control muscles. In the absence of nerves, the degree of regeneration in 21-day noninnervated EDL grafts was not significantly different between young and old rats. Similarly, when EDL muscles were damaged by Marcaine and regenerated in the presence of uninterrupted innervation, no differences were noted between young and old rats. These data support the working hypothesis that a deficiency in reinnervation with increasing age accounts, at least in part, for the poorer success of muscle regeneration in grafts in old compared with young rats.

Muscle regeneration in young and old rats: effects of motor nerve transection with and without marcaine treatment.

We tested the hypothesis that after skeletal muscle regeneration in old compared with young rats damage to the motor nerve rather than damage to muscle fibers determines the magnitude of the deficits in muscle mass and maximum force (Po). The mass and Po of extensor digitorum longus (EDL) muscles of young (4 months) and old (24 months) male rats were compared two months following (i) Marcaine treatment plus simultaneous motor nerve transection, (ii) motor nerve transection alone, and (iii) Marcaine treatment alone (from data compiled previously). In both the nerve transection-only and Marcaine with nerve transection groups the recovery of mass and Po was significantly greater in young than in old rats. This is in contrast to our previous data showing that in the absence of nerve damage Marcaine-treated muscle in old rats regenerates as well as that in young rats. Our hypothesis was supported, and we conclude that impaired axonal regeneration, re-establishment of nerve-muscle contact, or both, is the critical component in the impaired regeneration of muscle grafts in old as compared with young rats.

Skeletal muscle regeneration in very old rats.

This study was undertaken to assess the regenerative capacity of skeletal muscle in rats near the end of their normal life span. Two experiments were performed. In the first, extensor digitorum longus (EDL) muscles were cross-age transplanted from 32-month-old male inbred Wistar (WI/HicksCar) rats in place of an EDL muscle in 4-month-old hosts. The other EDL muscle in the hosts was autotransplanted. After 60 days, the old-into-young muscle transplants regenerated as well as the young-into-young autotransplants. In the second experiment, EDL muscles in young adult (4 months) and old rats (32 and 34 months) of WI/HicksCar and Brown Norway (BN) were injected with a local anesthetic, bupivacaine, and allowed to regenerate for 41 days. In all cases, the masses and absolute maximum tetanic force of the regenerates equaled or exceeded those of untouched contralateral control muscles. These experiments showed that under appropriate conditions, very old muscles can regenerate to equal or exceed the contralateral control values, which in old rats are much less than those in muscles of young rats.

Motor unit properties of nerve-intact extensor digitorum longus muscle grafts in young and old rats.

Impaired reinnervation has been implicated as the cause of the threefold disparity in the recovery of maximum force (P0) of standard muscle grafts in old compared with young rats. The specific, null hypothesis of this study is that compared with age-matched control extensor digitorum longus (EDL) muscles, nerve-intact EDL muscle grafts in young and old rats show no evidence of an age-related impairment in reinnervation. Nerve-intact grafts were performed in 3-month-old and 23-month-old rats and were evaluated 60 days postoperatively. Compared with age-matched control EDL muscles, nerve-intact grafts in young and old rats showed no difference in muscle mass or motor unit numbers. The mean motor unit P0 for nerve-intact graft muscles in both age groups was significantly lower than that of age-matched control muscles. These data support our hypothesis that if axons are allowed to regenerate in an endoneurial environment, there is no evidence of an age-related impairment in muscle reinnervation.

A histological study of local anesthetic-induced muscle degeneration and regeneration in the monkey.

Small amounts (1-2 ml) of local anesthetics (bupivacaine, lidocaine, and mepivacaine) were injected into the abductor pollicis brevis muscles of 35 monkeys. Control muscles were injected with saline. The muscles were preserved for histology from 4.5 h to 48 days after the injection. Histological damage to muscle fibers was evident from the time of the first sampling. Invasion of damaged muscle fibers by phagocytic cells was prominent by 2-3 days postinjection. At 4 to 5 days, areas of muscle fiber damage were characterized by dense concentrations of phagocytes and mononuclear myoblastic cells. At 6 days, fields of early myotubes were evident. Maturation of myotubes into immature cross-striated muscle fibers occurred over the next week. Occasional myotubes or immature regenerating muscle fibers were seen as late as 28 days. The topographical pattern of muscle fiber degeneration and regeneration showed a concentration along the surfaces of muscle fascicles or, if intrafascicular, around the presumed site of injection.

An electron microscopic study of local anesthetic-induced skeletal muscle fiber degeneration and regeneration in the monkey.

An electron microscopic study was done on abductor pollicis brevis muscles of 18 Rhesus monkeys after intramuscular injections of 0.75% bupivacaine, 2% mepivacaine, or 2% lidocaine + epinephrine. The muscles were examined for from 2 h to 28 days. Severe muscle fiber damage, consisting of breakdown of sarcolemma and myofibrils, was seen as early as 2 h. Phagocyte mediated fragmentation of the degenerating muscle fibers was at its peak during the third and fourth days. Myoblasts were abundant during the fourth day. Early myotubes appeared on the fifth and sixth days, and they matured during the second week. Satellite cells appeared alongside mature myotubes. Overall, the local anesthetic-induced breakdown and regeneration of skeletal muscle fibers in the monkey followed a course quite similar to that seen in the rat.

The recovery of long-term denervated rat muscles after Marcaine treatment and grafting.

Disruption of the nerve supply results in the rapid loss of mass and contractile force in skeletal muscles. These losses are reversible to a high degree in short-term denervated muscles with grafting and nerve implantation. However, return is much poorer in long-term denervated muscles. This study examined the basis for the differences in the recovery of non-denervated and 7-month denervated rat extensor digitorum longus (EDL) muscles after grafting and nerve implantation. We found that the level of recovery is related to the ability of muscle fibers to degenerate and regenerate after grafting. Fibers within long-term denervated muscles do not degenerate and regenerate as well as those within muscles which are not denervated prior to grafting. The functional recovery of the denervated muscles is significantly improved when their fibers are induced to degenerate with the myotoxic anesthetic, Marcaine, Degeneration of these fibers is followed by massive regeneration. The finding that denervated muscles are capable of being restored to a significant level by inducing regeneration may be useful in the clinical treatment of denervated muscles.

Cellular responses to free grafting of the extensor digitorum longus muscle of the rat.

The cellular and subcellular responses related to the survival or destruction and subsequent regeneration of muscle fibers within the freely grafted extensor digitorum longus muscle of the rat were examined by light and electron microscopy. A small number of fibers at the periphery of the grafts survived the initial ischemia but underwent denervation changes and accumulated lipid deposits. The majority of fibers in the grafts, however, became ischemic and underwent an intrinsic degeneration within 4 hours. Cell-mediated destruction of the degenerating fibers occurred as the grafts became revascularized. The basal laminae and some of the satellite cells were the only elements of the original fibers that persisted. Regeneration began at the periphery of the graft within three days after grafting and reached the center about three days later. After phagocytosis of the original fibers, presumptive myoblasts within the grafts differentiated into myoblasts and myotubes. The formation of myotubes followed a biphasic pattern of development comparable to that of normal fetal muscle. Although most of the myotubes were formed within the basal lamina remaining from the original fiber, there was also evidence for regeneration outside the basal lamina. Myotubes matured into muscle fibers which were essentially normal in apperance when examined up to 180 days after grafting. Some fibers, however, were atrophic, presumably due to a failure to become innervated, and some fibers were joined by myo-myous junctions. Pre-denervated grafts and Marcaine-treated grafts were also examined. There were more surviving fibers in pre-denervated grafts, and cell-mediated destruction of degenerating fibers proceeded more rapidly than in normal grafts. No surviving fibers were found in Marcaine-treated grafts. The changes in these grafts were otherwise similar to normal grafts. A schematic model of the spatial and temporal sequence of degeneration and regeneration within a free muscle graft is presented.

Developmental patterns of glycolytic enzymes in regenerating skeletal muscle after autogenous free grafting.

Extensor digitorum longus muscles of rats were removed and injected with a solution of Marcaine plus hyaluronidase. After incubation in Marcaine solution for 10 min, the muscles were grafted into their original beds. The grafts and the contralateral control muscles were removed from the rats at 0, 1-5, 7, 11, 36, and 69 days postoperatively. The muscles were then frozen in dry ice and isopentane and subsequently homogenized and centrifuged. The supernatant was analyzed for a number of enzymes, the regenerative patterns of which can be classified into 3 groups: (1) early increase in activity: hexokinase, glucose-6-phosphate dehydrogenase; (2) early decrease in activity with failure to recover to control levels: phosphorylase, phosphofructokinase, alpha-glycerophosphate dehydrogenase; and (3) early decrease followed by return to control levels: lactate dehydrogenase, pyruvate kinase, creatine phosphokinase, adenylate kinase. These patterns are not identical to those reported for embryogenesis of muscle. The data are discussed with regard to correlative histological studies of muscle regeneration.