Progesterone prevents mitochondrial dysfunction in the spinal cord of wobbler mice

In the Wobbler mouse, a mutation of the Vps54 protein increases oxidative stress in spinal motoneurons, associated to toxic levels of nitric oxide and hyperactivity of nitric oxide synthase (NOS). Progesterone neuroprotection has been reported for several CNS diseases, including the Wobbler mouse neurodegeneration. In the present study, we analyzed progesterone effects on mitochondrial-associated parameters of symptomatic Wobbler mice. The activities of mitochondrial respiratory chain complexes I, II-III and IV and protein levels of mitochondrial and cytosolic NOS were determined in cervical and lumbar cords from control, Wobbler and Wobbler mice receiving a progesterone implant for 18 days. We found a significant reduction of complex I and II-III activities in mitochondria and increased protein levels of mitochondrial, but not cytosolic nNOS, in the cervical cord of Wobbler mice. Progesterone treatment prevented the reduction of complex I in the cervical region and the increased level of mitochondrial nNOS. Wobbler motoneurons also showed accumulation of amyloid precursor protein immunoreactivity and decreased activity and immunostaining of MnSOD. Progesterone treatment avoided these abnormalities. Therefore, administration of progesterone to clinically afflicted Wobblers (i) prevented the abnormal increase of mitochondrial nNOS and normalized respiratory complex I; (ii) decreased amyloid precursor protein accumulation, a sign of axonal degeneration, and (iii) increased superoxide dismutation. Thus, progesterone neuroprotection decreases mitochondriopathy of Wobbler mouse cervical spinal cord.     

Basis of progesterone protection in spinal cord neurodegeneration

Progesterone neuroprotection has been reported in experimental brain, peripheral nerve and spinal cord injury. To investigate for a similar role in neurodegeneration, we studied progesterone effects in the Wobbler mouse, a mutant presenting severe motoneuron degeneration and astrogliosis of the spinal cord. Implant of a single progesterone pellet (20 mg) during 15 days produced substantial changes in Wobbler mice spinal cord. Morphologically, motoneurons of untreated Wobbler mice showed severe vacuolation of intracellular organelles including mitochondria. In contrast, neuropathology was less pronounced in Wobbler mice receiving progesterone, together with a reduction of vacuolated cells and preservation of mitochondrial ultrastructure. Determination of mRNAs for the 3 and β1 subunits of neuronal Na, K-ATPase, showed that mRNA levels in untreated mice were significantly reduced, whereas progesterone therapy re-established the expression of both subunits. Additionally, progesterone treatment of Wobbler mice attenuated the aberrant expression of the growth-associated protein (GAP-43) mRNA which otherwise occurred in motoneurons of untreated animals. The hormone, however, was without effect on astrocytosis of Wobbler mice, determined by glial fibrillary acidic protein (GFAP)-immunostaining. Lastly, progesterone treatment of Wobbler mice enhanced grip strength and prolonged survival at the end of the 15-day observation period. Recovery of morphology and molecular motoneuron parameters of Wobbler mice receiving progesterone, suggest a new and important role for this hormone in the prevention of spinal cord neurodegenerative disorders.     

Cellular basis of steroid neuroprotection in the wobbler mouse, a genetic model of motoneuron disease

1. The Wobbler mouse suffers an autosomal recessive mutation producing severe motoneuron degeneration and astrogliosis in the spinal cord. It has been considered a suitable model of human motoneuron disease, including the sporadic form of amyotrophic lateral sclerosis (ALS).2. Evidences exist demonstrating increased oxidative stress in the spinal cord of Wobbler mice, whereas antioxidant therapy delayed neurodegeneration and improved muscle trophism. 21-Aminosteroids are glucocorticoid-derived hydrophobic compounds with antioxidant potency 3 times higher than vitamin E and 100 times higher than methylprednisolone. They do not bind to intracellular receptors, and prevent lipid peroxidation by insertion into membrane lipid bilayers.3. In common with the spinal cord of ALS patients, Wobbler mice present astrocytosis with hyperexpression of glial fibrillary acidic protein (GFAP), and increased expression of nitric oxide synthase (NOS) and growth-associated protein (GAP-43) in motoneurons. Here, we review our studies on the effects of a 21-aminosteroid on GFAP, NOS, and GAP-43.4. First, we showed that 21-aminosteroid treatment further increased GFAP-expressing astrocytes in gray matter of the Wobbler spinal cord. This effect may provide neuroprotection if one considers a trophic and beneficial function of astrocytes during the course of degeneration. Other neuroprotectans used in Wobbler mice (T-588) also increased preexisting astrocytosis.5. Second, histochemical determination of NADPH-diaphorase, a parameter indicative of neuronal NOS activity, showed that the 21-aminosteroid down-regulated the high activity of this enzyme in ventral horn motoneurons. Therefore, suppression of nitric oxide by decreasing NADPH-diaphorase (NOS) activity may provide neuroprotection considering that excess NO is highly toxic to motoneurons.6. Finally, 21-aminosteroid treatment significantly attenuated the aberrant expression of both GAP-43 protein and mRNA in Wobbler motoneurons. Hyperexpression of GAP-43 possibly indicated abnormal synaptogenesis, denervation, and muscle atrophy, parameters which may return to normal following antioxidant steroid treatment.7. Besides 21-aminosteroids, other steroids also behave as neuroprotectans. In this regard, degenerative diseases may constitute potential targets of these hormones, based on the fact that the spinal cord expresses in a regional and cell-specific fashion, receptors for androgens, progesterone, adrenal steroids, and estrogens.     

Progesterone Effects on Neuronal Ultrastructure and Expression of Microtubule-associated Protein 2 (MAP2) in Rats with Acute Spinal Cord Injury

(1) Following acute spinal cord injury, progesterone modulates several molecules essential for motoneuron function, although the morphological substrates for these effects are unknown. (2) The present study analyzed morphological changes in motoneurons distal to the lesion site from rats with or without progesterone treatment. We employed electron microscopy to study changes in nucleus and cytoplasm and immunohistochemistry for the microtubule-associated protein 2 (MAP2) for changes in cytoskeleton. (3) After spinal cord injury, the nucleoplasm appeared more finely dispersed resulting in reduced electron opacity and the nucleus adopted an eccentric position. Changes of perikarya included dissolution of Nissl bodies and dissociation of polyribosomes (chromatolysis). After progesterone treatment for 3 days, the deafferented motoneurons now presented a clumped nucleoplasm, a better-preserved rough endoplasmic reticulum and absence of chromatolysis. Progesterone partially prevented development of nuclear eccentricity. Whereas 50% of injured motoneurons showed nuclear eccentricity, only 16% presented this phenotype after receiving progesterone. Additionally, injured rats showed reduced immunostaining for MAP2 in dendrites, pointing to cytoskeleton abnormalities, whereas progesterone treatment attenuated the injury-induced loss of MAP2. (4) Our data indicated that progesterone maintained in part neuronal ultrastructure, attenuated chromatolysis, and preclude the loss of MAP2, suggesting a protective effect during the early phases of spinal cord injury.     

The 21-Aminosteroid U-74389F Attenuates Hyperexpression of GAP-43 and NADPH-Diaphorase in the Spinal Cord of Wobbler Mouse,a Model for Amyotrophic Lateral Sclerosis

The wobbler mouse suffers an autosomal recessive mutation producing severe neurodegeneration and astrogliosis in spinal cord. It has been considered a model for amyotrophic lateral sclerosis. We have studied in these animals the expression of two proteins, the growth-associated protein (GAP-43) and the NADPH-diaphorase, the nitric oxide synthesizing enzyme, employing immunocytochemistry and histochemistry. We found higher expression of GAP-43 immunoreactivity in dorsal horn, Lamina X, corticospinal tract and ventral horn motoneurons in wobbler mice compared to controls. Weak NADPH-diaphorase activity was present in control motoneurons, in contrast to intense labeling of the wobbler group. No differences in diaphorase activity was measured in the rest of the spinal cord between control and mutant mice. A group of animals received subcutaneously for 4 days a 50 mg pellet of U-74389F, a glucocorticoid-derived 21-aminosteroid with antioxidant properties but without glucocorticoid activity. U-74389F slightly attenuated GAP-43 immunostaining in dorsal regions of the spinal cord from wobblers but not in controls. However, in motoneurons of wobbler mice number of GAP-43 immunopositive neurons, cell processes and reaction intensity were reduced by U-74389F. The aminosteroid reduced by 50% motoneuron NADPH-diaphorase activity. Hyperexpression of GAP-43 immunoreactivity in wobbler mice may represent an exaggerated neuronal response to advancing degeneration or muscle denervation. It may also be linked to increased nitric oxide levels. U-74389F may stop neurodegeneration and/or increase muscle trophism and stop oxidative stress, consequently GAP-43 hyperexpression was attenuated. Wobbler mice may be important models to evaluate the use of antioxidant steroid therapy with a view to its use in human motoneuron disease.     

Cholinergic differentiation factor (CDF/LIF) promotes survival of isolated rat embryonic motoneurons in vitro.

We present evidence that the cholinergic differentiation factor (CDF), originally purified from cardiac and skeletal muscle cell-conditioned medium and found to be identical to leukemia inhibitory factor (LIF), promotes survival of embryonic day 14 rat motoneurons in vitro. These neurons were retrogradely labeled with the fluorescent tracer Dil and enriched on a density gradient or purified to homogeneity by fluorescence-activated cell sorting. Subnanomolar concentrations of CDF/LIF supported the survival of 85% of the motoneurons that would have died between days 1 and 4 of culture. The enhanced survival was accompanied by a 4-fold increase in choline acetyltransferase (ChAT) activity per culture. CDF/LIF also increased ChAT activity in dorsal spinal cord cultures, but had no detectable effect on ChAT levels in septal or striatal neuronal cultures. For comparison, other neurotrophic molecules were tested on motoneuron cultures. Ciliary neurotrophic factor had effects on motoneuron survival similar to those of CDF/LIF, whereas basic fibroblast growth factor was somewhat less effective. Nerve growth factor had no effect on the survival of rat motoneurons.     

Calpain Inhibition Protects Spinal Motoneurons from the Excitotoxic Effects of AMPA In vivo

Microdialysis perfusion of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in rat lumbar spinal cord produces severe motoneuron damage and consequently hindlimb paralysis. Here we studied the time course of the AMPA-induced neurodegenerative changes and motor alterations, and the protective effect of leupeptin, an inhibitor of calpain, a Ca(2+)-activated protease. Paralysis occurs at 4-6 h after AMPA perfusion, but cresyl violet staining showed that motoneuron damage starts at about 3 h and progresses until reaching 50% neuronal loss at 6 h and 90% loss at 12 h. In contrast, choline acetyltransferase (ChAT) immunohistochemistry revealed that the enzyme is already decreased at 30 min after AMPA perfusion and practically disappears at 3 h. Microdialysis coperfusion of leupeptin with AMPA prevented the motor alterations and paralysis and remarkably reduced both the decrement in ChAT immunoreactivity and the loss of motoneurons. We conclude that an increased Ca(2+) influx through Ca(2+)-permeable AMPA receptors activates calpain, and as a consequence ChAT content decreases earlier than other Ca(2+)-dependent processes, including the proteolytic activity of calpain, cause the death of motoneurons.     

Analysis of impulse adaptation in motoneurons

Animal locomotion results from muscle contraction and relaxation cycles that are generated within the central nervous system and then are relayed to the periphery by motoneurons. Thus, motoneuron function is an essential element for understanding control of animal locomotion. This paper presents motoneuron input–output relationships, including impulse adaptation, in the medicinal leech. We found that although frequency-current graphs generated by passing 1-s current pulses in neuron somata were non-linear, peak and steady-state graphs of frequency against membrane potential were linear, with slopes of 5.2 and 2.9 Hz/mV, respectively. Systems analysis of impulse frequency adaptation revealed a static threshold nonlinearity at −43 mV (impulse threshold) and a single time constant (τ = 88 ms). This simple model accurately predicts motoneuron impulse frequency when tested by intracellular injection of sinusoidal current. We investigated electrical coupling within motoneurons by modeling these as three-compartment structures. This model, combined with the membrane potential–impulse frequency relationship, accurately predicted motoneuron impulse frequency from intracellular records of soma potentials obtained during fictive swimming. A corollary result was that the product of soma-to-neurite and neurite-to-soma coupling coefficients in leech motoneurons is large, 0.85, implying that the soma and neurite are electrically compact.     

Rescue of motoneurons from cell death by a purified skeletal muscle polypeptide: effects of the ChAT development factor, CDF

Rat skeletal muscle contains a 22 kd polypeptide that increases the level of choline acetyltransferase (ChAT) activity in cultures of embryonic rat spinal cord neurons and has been purified to homogeneity. The application of this factor, ChAT development factor or CDF, to developing chick embryos during the period of naturally occurring motoneuron cell death significantly increased the survival of motoneurons but did not affect the survival of dorsal root ganglion neurons or sympathetic preganglionic neurons (column of Terni). These results provide the first demonstration that an isolated, skeletal muscle-derived molecule can selectively enhance the survival of motoneurons in vivo and suggest that CDF may function in vivo to regulate the survival and development of motoneurons.     

Insulin-like growth factors: Putative muscle-derived trophic agents that promote motoneuron survival

Treatment of chick embryos in ovo with IGF-I during the period of normal, developmentally regulated neuronal death (embryonic days 5–10) resulted in a dose-dependent rescue of a significant number of lumbar motoneurons from degeneration and death. IGF-II and two variants of IGF-I with reduced affinity for IGF binding proteins, des(1-3) IGF-I and long R³ IGF-I, also elicited enhanced survival of motoneurons equal to that seen in IGF-I-treated embryos. IGF-I did not enhance mitogenic activity in motoneuronal populations when applied to embryos during the period of normal neuronal proliferation (E2-5). Treatment of embryos with IGF-I also reduced two types of injury-induced neuronal death. Following either deafferentation or axotomy, treatment of embryos with IGF-I rescued approximately 75% and 50%, respectively, of the motoneurons that die in control embryos as a result of these procedures. Consistent with the survival-promoting activity on motoneurons in ovo, IGF-I, -II, and des(1-3) IGF-I elevated choline acetyltransferase activity in embryonic rat spinal cord cultures, with des (1-3) IGF-I demonstrating 2.5 times greater potency than did IGF-I. A single addition of IGF-I at culture initiation resulted in the maintenance of 80% of the initial ChAT activity for up to 5 days, during which time ChAT activity in untreated control cultures fell to 9%. In summary, these results demonstrate clear motoneuronal trophic activity for the IGFs. These findings, together with previous reports that IGFs are synthesized in muscle and may participate in motoneuron axonal regeneration and sprouting, indicate that these growth factors may have an important role in motoneuron development, maintenance, and recovery from injury. © 1993 John Wiley & Sons, Inc.     

Modifications of motoneuron development following transplantation of thoracic spinal cord to the lumbar region in the chick embryo: Evidence for target-derived signals that regulate differentiation

In order to examine the role of target cells in the development of spinal motoneurons, the neural tube from thoracic segments was transplanted to the lumbar region on embryonic day (E) 2, and allowed to innervate hindlimb muscles in the chick embryo. When examined at later stages of development, the proportion of white and gray matter in the thoracic transplant was altered to resemble normal lumbar cord. Many thoracic motoneurons were able to survive up to posthatching stages following transplantation. The branching and arborization of dendrites of thoracic motoneurons innervating hindlimb muscles, as well as motoneuron (soma) size, were also increased to an extent approximating that seen in normal lumbar motoneurons. In support of previous studies using a similar transplant model, we have also found that the peripheral (intramuscular) branching pattern of thoracic motoneuron axons innervating hindlimb muscles was similar to that of normal lumbar motoneurons. Axon size and the degree of myelination of transplanted thoracic motoneuron axons were also increased so that these parameters more closely resembled axons of normal lumbar than normal thoracic spinal motoneurons. Virtually all of the changes in motoneuron properties noted above were observed irrespective of whether or not the transplanted spinal cord had developed in anatomical continuity with the host rostral cord. Accordingly, it is unlikely that the changes in the development of transplanted thoracic motoneurons reported here are induced either entirely, or in part, by signals derived from the host central nervous system. Rather, these changes appear to be mediated by interactions between the transplanted motoneurons and the hindlimb. We favor the notion that retrograde trophic signals derived from the hindlimb act to modulate the development of innervating motoneurons. Whether this signal involves a diffusible trophic agent released from target cells, or acts by some other mechanism is presently unknown. © 1992 John Wiley & Sons, Inc.     

Elucidating the molecular mechanisms that underlie the target control of motoneuron death

Approximately half of the motoneurons generated during normal embryonic development undergo programmed cell death. Most of this death occurs during the time when synaptic connections are being formed between motoneurons and their target, skeletal muscle. Subsequent muscle activity stemming from this connection helps determine the final number of surviving motoneurons. These observations have given rise to the idea that motoneuron survival is dependent upon access to muscle derived trophic factors, presumably through intact neuromuscular synapses. However, it is not yet understood how the muscle regulates the supply of such trophic factors, or if there are additional mechanisms operating to control the fate of the innervating motoneuron. Recent observations have highlighted target independent mechanisms that also operate to support the survival of motoneurons, such as early trophic-independent periods of motoneuron death, trophic factors derived from Schwann cells and selection of motoneurons during pathfinding. Here we review recent investigations into motoneuron cell death when the molecular signalling between motoneurons and muscle has been genetically disrupted. From these studies, we suggest that in addition to trophic factors from muscle and/or Schwann cells, specific adhesive interactions between motoneurons and muscle are needed to regulate motoneuron survival. Such interactions, along with intact synaptic basal lamina, may help to regulate the supply and presentation of trophic factors to motoneurons.     

Relevance of motoneuron specification and programmed cell death in embryos to therapy of ALS

The molecular cues that generate spinal motoneurons in early embryonic development are well defined. Motoneurons are generated in excess and consequently undergo a natural period of programmed cell death. Although it is not known exactly how motoneurons compete for survival in embryonic development, it is hypothesized that they rely on the ability to access limited amounts of trophic factors from peripheral tissues, a process that is tightly regulated by skeletal muscle activity. Attempts to elucidate the molecular mechanisms that underlie motoneuron generation and programmed cell death in embryos have led to various effective strategies for treating injury and disease in animal models. Such studies provide great hope for the amelioration of human amyotrophic lateral sclerosis (ALS), a devastating progressive motoneuron degenerative disease. Here we review the clinical relevance of studying motoneuron specification and death during embryonic development.     

Postembryonic development of centrally generated flight motor patterns in the hawkmoth, Manduca sexta

This study analyses the maturation of centrally generated flight motor patterns during metamorphosis of Manduca sexta. Bath application of the octopamine agonist chlordimeform to the isolated central nervous system of adult moths reliably induces fictive flight patterns in wing depressor and elevator motoneurons. Pattern maturation is investigated by chlordimeform application at different developmental stages. Chlordimeform also induces motor patterns in larval ganglia, which differ from fictive flight, indicating that in larvae and adults, octopamine affects different networks. First changes in motoneuron activity occur at the pupal stage P10. Rhythmic motor output is induced in depressor, but not in elevator motoneurons at P12. Adult-like fictive flight activity in motoneurons is observed at P16 and increases in speed and precision until emergence 2 days later. Pharmacological block of chloride channels with picrotoxin also induces fictive flight in adults, suggesting that the pattern-generating network can be activated by the removal of inhibition, and that proper network function does not rely on GABA_A receptors. Our results suggest that the flight pattern-generating network becomes gradually established between P12 and P16, and is further refined until adulthood. These findings are discussed in the context of known physiological and structural CNS development during Manduca metamorphosis.     

A morphometric analysis of the spinal motor pool in relation to its target muscle during growth in the trout, Salmo gairdneri Richardson

The relation between number and size of spinal motoneurons and the dimension of myotomal muscle has been investigated in trout at different stages of embryonic, larval and postlarval development (body length 1–15 cm). Three spinal segments have been analysed (cervical, trunk and caudal) and the following parameters were determined by means of a Micromeasurements Image Analyzer: (a) mean cross-sectional myotomal area; (b) mean soma size of principal (or dorsomedian, DM) and secondary (or ventrolateral, VL) motoneurons; (c) DM and VL motoneuron density per segment. Myotomal muscle and motor pool growth was evaluated by percent increments of a, b and c parameters at each stage. Their relationships were denned by equations of computed regression lines.
The analysis provided evidence that: (1) a continuous exponential growth of mean myotomal area takes place in the three segments, with the same trend and with the lowest values in the caudal segment; (2) DM and VL motoneuron size and density per segment also increase during development, with the least value in the caudal segment, VL parameters being of lesser value than DM; (3) motoneuron pool and its target myotomal muscle parameters bear a linear relationship as defined by equations of computer regression lines; (4) motoneuron number percent increment at early eleutherembryonic stage precedes myotomal area increment which takes place during late eleutherembryonic stage.
It is apparent that spinal motor pool and target myotomal muscle grow at the same rate in the trout during the considered stages. The discussion links this fact with the hypothesis of a neuronal influence on muscle fibre type differentiation.     

Monosynaptic control of spinal motoneurons from different levels of the brain

In experiments on cats and monkeys it is established that reticulo-, rubro-, and corticomotoneuronal influences are characterized by a number of common features: 1) they are produced by fast conducting fibers of the descending tracts; 2) they do not attain the critical level needed for AP generation; and 3) they are caused by implication of synapses that are predominantly located on dendrites of the motoneurons. Results of experiments carried out on lampreys and rats indicate that reticulo-motoneuronal monosynaptic projections emerge already at the earliest stages of vertebrate evolution and retain their significance in mammals. The data of research on supraspinal influences during ontogenesis indicate early development of descending stem projections. This enables us to regard cerebro-motoneuronal monosynaptic connections as an important component of supraspinal control of motoneurons, a component whose functional role is in large measure determined by interaction with other synaptic inputs of the motoneuron.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 2, pp. 203–215, March–April, 1970.     

Retinaldehyde dehydrogenase 2 and Hoxc8 are required in the murine brachial spinal cord for the specification of Lim1+ motoneurons and the correct distribution of Islet1+ motoneurons

Retinoic acid (RA) activity plays sequential roles during the development of the ventral spinal cord. Here, we have investigated the functions of local RA synthesis in the process of motoneuron specification and early differentiation using a conditional knockout strategy that ablates the function of the retinaldehyde dehydrogenase 2 (Raldh2) synthesizing enzyme essentially in brachial motoneurons, and later in mesenchymal cells at the base of the forelimb. Mutant (Raldh2L-/-) embryos display an early embryonic loss of a subset of Lim1+ brachial motoneurons, a mispositioning of Islet1+ neurons and inappropriate axonal projections of one of the nerves innervating extensor limb muscles, which lead to an adult forepaw neuromuscular defect. The molecular basis of the Raldh2L-/- phenotype relies in part on the deregulation of Hoxc8, which in turn regulates the RA receptor RARbeta. We further show that Hoxc8 mutant mice, which exhibit a similar congenital forepaw defect, display at embryonic stages molecular defects that phenocopy the Raldh2L-/- motoneuron abnormalities. Thus, interdependent RA signaling and Hox gene functions are required for the specification of brachial motoneurons in the mouse.     

Nutritional status influences reproductive seasonality in Creole goats: 1. Ovarian activity during seasonal reproductive transitions

The objective was to determine the effect of body energy stores, evaluated by a body mass index (BMI), and food intake (FI), on the length of the anovulatory period and ovarian activity during the seasonal reproductive transitions in Creole goats. Non-pregnant, non-lactating Creole goats (n = 28) were fed to induce two different BMI conditions: Greater (GBMI; n = 15), and Lesser (LBMI; n = 13). Each BMI group was divided into two sub-groups, which were either feed restricted (FR) or non-feed restricted (NFR). Goats in the NFR groups received a diet containing 100% of the daily maintenance requirements (basal diet), while restricted goats were subjected to alternated periods, receiving 100% (11 d) and 60% (10 d) of the basal diet, during the entire experimental period. The experiment started after does were treated to synchronize time of estrus. Serum progesterone was determined in samples obtained twice a week, and used as a criterion for determining ovulations. During the transition to the anovulatory period three transrectal ovarian ultrasonographic scans were performed in a sub-group of 12 goats (n = 3 for each treatment combination). The diameter of the largest follicle (LFD) and the total number of antral follicles ≥2 mm (TAF) were recorded. Ultrasonographic ovarian scans were performed at 21, 42 and 63 days after the beginning of the experiment, concurrently with the end of each feed restriction period. The variables of response associated with ovulation were not influenced by BMI or BMI × FI interaction. However, FI influenced length of anovulatory season, as the anovulatory period was 30 d longer (P < 0.05) in the FR group as compared with the NFR group. Independently of treatments, TAF and LFD decreased from the first to the third ultrasonographic ovarian scan (13.2, 10.8 and 4.4 follicles; 3.7, 2.7 and 2.3 mm). Nevertheless, in PER 1 the number of TAF was greater (P < 0.05) in the FR as compared with NFR group and the GBMI group had a larger LFD (P < 0.05) as compared to the LBMI group. It is concluded, that temporal restriction in feed intake could affect the time of cessation and initiation of ovulations during the periods of transition to seasonal anestrus and return to estrous activity, and increase the length of the anovulatory period. In addition, ovarian follicular development during transition into the anovulatory period is differentially influenced by food intake and the status of body energy stores.     

Longitudinal neuronal organization and coordination in a simple vertebrate: a continuous,semi-quantitative computer model of the central pattern generator for swimming in young frog tadpoles

When frog tadpoles hatch their swimming requires co-ordinated contractions of trunk muscles, driven by motoneurons and controlled by a Central Pattern Generator (CPG). To study this co-ordination we used a 3.5 mm long population model of the young tadpole CPG with continuous distributions of neurons and axon lengths as estimated anatomically. We found that: (1) alternating swimming-type activity fails to self-sustain unless some excitatory interneurons have ascending axons, (2) a rostro-caudal (R-C) gradient in the distribution of excitatory premotor interneurons with short axons is required to obtain the R-C gradient in excitation and resulting progression of motoneuron firing necessary for forward swimming, (3) R-C delays in motoneuron firing decrease if excitatory motoneuron to premotor interneuron synapses are present, (4) these feedback connections and the electrical synapses between motoneurons synchronise motoneuron discharges locally, (5) the above findings are independent of the detailed membrane properties of neurons.