The role of Ca2+ in the elimination of polyneuronal innervation of rat soleus muscle fibres

The mechanism by which nerve - muscle contacts are reduced during postnatal development of the rat soleus muscle was investigated using electrophysiological methods. Between days 7 and 9 after birth, soleus muscle fibres lose 0.19–0.24 terminals per muscle fibre within 24 h. A much more rapid loss of contacts is seen when muscles are exposed in vitro to acetylcholine (10⁻³ g/ml). In this case 0.67–0.87 terminals per muscle fibre lose contact within 2 h. The loss of neuromuscular contacts induced by acetylcholine can be reduced by preincubating the muscles in solutions containing acetoxymethyl ester of 1,2-bis(2-amino-phenoxylethane-N,N₁;N₁-tetraacetic acid (BAPTA-AM), a Ca²⁺ chelating agent that enters cells and reduces the Ca²⁺ transients inside the cell. Treatment of muscles with nifedipine, which blocks dihydropyridine-sensitive (L-type) Ca²⁺ channels, also reduced the acetylcholinesterase-induced loss of neuromuscular contacts. The results indicate that transient increases in Ca²⁺ inside nerve terminals contribute to loss of neuromuscular contacts, and that these increases occur by Ca²⁺ entry through L-type channels.     

Non-quantal Release of Acetylcholine Affects Polyneuronal Innervation on Developing Rat Muscle Fibres

The membrane potential at endplates of the rat hemidiaphragm for 9-day-old rats increases by 1.8 mV after addition of D-tubocurarine. The endplate depolarization before the addition of D-tubocurarine is considered to be due to non-quantal release (NQR) of acetylcholine (ACh). In the presence of an anticholinesterase this depolarization increased. It was further enhanced by 0.1 -1.0 mM Mg²⁺ and reduced by 4 mM Mg²⁺ concentration. Thus the regulation of NQR at neuromuscular junctions of developing rat muscles is similar to that seen in adult mammalian species. The effect of NQR of ACh on neuromuscular contacts of muscle fibres from 8–9-day-old rat diaphragm and soleus muscles was studied. Pre-incubating the muscles in solutions where NQR was increased by lowering Mg²⁺ caused a significant ( P < 0.01) reduction of neuromuscular contacts. This reduction did not occur when muscles were incubated in high Mg²⁺, when NQR is reduced. Increasing quantal release by high Ca²⁺ also caused a reduction of neuromuscular contacts. Histological examination of soleus muscle fibres treated with an anticholinesterase showed that muscles incubated in solutions with low (0.1 mM) concentrations of Mg²⁺ had significantly fewer neuromuscular contacts (38%) than those incubated in high concentrations of Mg²⁺ (61%). It is concluded that the NQR as assessed here contributes to the elimination of polyneuronal innervation during postnatal development of rat muscles.     

The Pontine A5 Noradrenergic Cells which Project to the Spinal Cord Dorsal Horn are Reciprocally Connected with the Caudal Ventrolateral Medulla in the Rat

A disynaptic pathway linking the caudal ventrolateral medulla (VLM) to the spinal cord via the A₅ noradrenergic cell group of the pons has recently been described in the rat. In the present work, the projections of the A₅ to the VLM and to the spinal dorsal horn were studied with double-tracing techniques combined with immunostaining of the noradrenaline-synthesizing enzyme dopamine-β-hydroxylase. Cholera toxin subunit B (CTb) injected into the VLM and fluoro-gold injected into the spinal dorsal horn produced double retrograde labelling of A₅ neurons immunoreactive for dopamine-β-hydroxylase, which received appositions of fibre varicosities labelled anterogradely with CTb injected into the VLM. After injecting CTb into the A₅, retrogradely labelled neurons occurred in the VLM. These neurons were contacted by anterogradely labelled fibres from the A₅ group. These observations indicate that the VLM cells acting upon the A₅ spinally projecting neurons, which are likely to exert an α₂-adrenoreceptor-mediated inhibition on the spinal cord, are targeted by collaterals of the A₅ spinal cord-bound axons. The A₅-VLM pathway may be the anatomical substrate of a negative feedback circuit whereby the modulatory action of the VLM on the spinal cord is self-inhibited through activation of the A₅.     

Blockade of A2a Adenosine Receptors Positively Modulates Turning Behaviour and c-Fos Expression Induced by D1 Agonists in Dopamine-denervated Rats

In rats with unilateral 6-hydroxydopamine lesions of the dopaminergic nigrostriatal pathway, administration of the A_2a adenosine antagonist SCH 58261 alone did not induce any motor asymmetry but strongly potentiated the contralateral turning behaviour induced by the dopamine D₁ agonist SKF 38393. SCH 58261 also increased the number of Fos-like positive nuclei induced by SKF 38393 in the 6-hydroxydopamine-lesioned striatum. Intense potentiation of D₁-dependent turning behaviour and c-Fos expression was also observed after administration of the A_2a/A₁ antagonist CGS 15943. Administration of the A₁ adenosine receptor antagonist DPCPX induced a small potentiation of D₁-mediated contralateral turning while c-Fos expression induced by SKF 38393 was not modified. The results suggest that endogenous adenosine acting on A_2a receptors can exert an inhibitory influence on the functional expression of D₁-mediated responses in dopamine-denervated rats, and propose new possible therapeutic approaches in the treatment of Parkinson's disease.     

Rat Colonic Motor Responses to Inflammatory Mediators In Vitro: A Poor Model for the Human Colon

There is evidence that the rat colon exposed to trinitrobenzenesulfonic acid (TNBS) develops a colitis-like inflammation which may be a model for inflammatory bowel disease (IBD) in humans. The aim of this study was to determine if the pharmacologic responses of the normal rat distal colon to several IBD-associated inflammatory mediators were sufficiently similar to those of the normal human colon for the rat with TNBS-induced colitis to provide an appropriate model for their effects on human colonic motility in IBD. Longitudinal and circular muscle mechanical properties were also investigated. Longitudinal muscle responded with concentration-dependent contractions to histamine, leukotriene D₄, and prostaglandins E₂ and F-₂α, which were mediated by a direct action. In general, circular muscle responses to these mediators were smaller, and in the cases of leukotriene D₄ and prostaglandin F₂α, these were modulated by the concurrent activation of intrinsic inhibitory neurons. The mechanical properties of rat distal colonic longitudinal and circular muscle differed significantly from the equivalent human muscles. In most aspects studied normal rat distal colonic smooth muscles were at variance with previously reported properties of the equivalent human muscles. Thus the rat colon would be inappropriate as a model to study potential inflammatory mediator-induced alterations of colonic motility in IBD.     

Dopamine D1 Receptor-mediated Facilitation of GABAergic Neurotransmission in the Rat Strioentopeduncular Pathway and its Modulation by Adenosine A1 Receptor-mediated Mechanisms

By using in vivo microdialysis it was found that one of the main functions of striatal dopamine D₁ receptors is to selectively facilitate GABAergic neurotransmission in the 'direct'strioentopeduncular pathway. D₁ receptors localized in the entopeduncular nucleus were also found to facilitate GABA release. However, results obtained from in vivo microdialysis, in vivo electrochemistry, immunohistochemistry and confocal laser microscopy suggested that entopeduncular D₁ receptors could only be activated under pharmacological conditions. Adenosine A₁ receptors were found to antagonistically modulate the D₁-mediated regulation of the strioentopeduncular pathway. Furthermore, using in situ hybridization D₁ and A₁ receptors were shown to be colocalized in medium-sized striatal neurons. These results show that the strioentopeduncular neuron is a main locus for adenosine-dopamine interactions in the brain.     

Up-regulation of adenosine A1 receptors in frontal cortex from Pick's disease cases

The adenosine A₁ receptor (A₁R)–adenylyl cyclase (AC) pathway was studied in post-mortem human frontal and occipital cortex from Pick's disease (PiD) cases and age-matched nondemented controls. In frontal cortex, the main brain area affected in PiD, A₁Rs, determined by radioligand binding, Western blotting and real-time PCR assays, were significantly increased in PiD samples, suggesting up-regulation of this receptor. AC activity was determined in basal and stimulated conditions via stimulatory guanine nucleotide binding proteins (Gs) using GTP, or directly with forskolin. Basal AC activity was reduced in brains from PiD cases. This agrees with the decrease in AC type I (AC I) level detected by Western blotting. However, inhibition of forskolin-stimulated AC activity by a selective A₁R agonist was significantly increased in brains from PiD. In occipital cortex, adenosine A₁R numbers were similar in control and PiD cases, and no significant differences were found in A₁R-mediated AC inhibition. These results show that the adenosine A₁R–AC transduction pathway is specifically up-regulated and sensitized in frontal cortex brain in PiD.     

The antiepileptogenic effect of electrical stimulation at different low frequencies is accompanied with change in adenosine receptors gene expression in rats

Purpose:   Previous studies have shown that the anticonvulsant effects of low-frequency stimulation (LFS) can be affected by activation of adenosine receptors. In the present study, the effect of LFS at different frequencies on kindling rate and adenosine receptors gene expression was investigated.
Methods:   Animals were kindled by perforant path stimulation in a rapid kindling manner. LFS (0.5, 1, and 5 Hz) was applied after termination of each kindling stimulation. Seizure severity was measured according to behavioral and electrophysiologic parameters. At the end of the experiments, adenosine A₁ and A_2A receptor gene expression were measured.
Results:   The inhibitory effect of LFS on kindling acquisition was observed at all frequencies. In addition, the inhibitory action of LFS on enhancement of field excitatory postsynaptic potential slope and population spike amplitude during kindling acquisition was not affected by the LFS frequency. However, the effects of LFS on paired-pulse recordings were greater at frequency of 5 Hz. Application of LFS during kindling acquisition also prevented the kindling induced decrease in the A₁ receptor gene expression and attenuated the level of A_2A receptor gene expression in the dentate gyrus. These effects were also greater at the frequency of 5 Hz.
Discussion:   According to these data, it may be suggested that the antiepileptogenic effects of LFS, developed through inhibition of synaptic transmission in the dentate gyrus, is mediated somehow through preventing the decrease of A₁ receptor and through attenuating the A_2A receptor gene expression. These effects might be dependent on the frequency of LFS.     

Acetylcholinesterase molecular forms in C57BL/6J dystrophic mice

Acetylcholinesterase (AChE) was extracted from normal and dystrophic C57BL/6J mouse hindlimb muscles and its molecular forms fractionated by sucrose density gradient ultracentrifugation. In the soleus muscles from 6- to 7-week-old mice an increase in the 3 Svedberg unit (S) and a decrease in the 16S AChE molecular forms was observed in dystrophic animals compared to controls. At 12-13 weeks of age, no major significant differences in the relative proportions of AChE molecular forms were noted. In the extensor digitorum longus (EDL) muscles of 6- to 7-week-old dystrophic mice a significant decrease in the proportion of the 10S AChE molecular form and an increase in the 3S and 5S forms was observed. At 12-13 weeks, the dystrophic EDL muscles again displayed a decrease in the 10S form; however, the increase in the 3S and 5S AChE forms, while still apparent, was not significant. These results provide evidence for a biochemical abnormality in the distribution of specific AChE molecular forms, and a differential expression of this abnormality in the soleus and EDL muscles.     

Specific impulse patterns regulate acetylcholinesterase activity in skeletal muscles of rats and rabbits

In rats, acetylcholinesterase (AChE) activity in the fast muscles is several times higher than in the slow soleus muscle. The hypothesis that specific neural impulse patterns in fast or slow muscles are responsible for different AChE activities was tested by altering the neural activation pattern in the fast extensor digitorum longus (EDL) muscle by chronic low-frequency stimulation of its nerve. In addition, the soleus muscle was examined after hind limb immobilization, which changed its neural activation pattern from tonic to phasic. Myosin heavy-chain (MHC) isoforms were analyzed by gel electrophoresis. Activity of the molecular forms of AChE was determined by velocity sedimentation. Low-frequency stimulation of the rat EDL for 35 days shifted the profile of MHC II isoforms toward a slower MHCIIa isoform. Activity of the globular G₁ and G₄ molecular forms of AChE decreased by a factor of 4 and 10, respectively, and became comparable with those in the soleus muscle. After hind limb immobilization, the fast MHCIId isoform, which is not normally present, appeared in the soleus muscle. Activity of the globular G₁ form of AChE increased approximately three times and approached the levels in the fast EDL muscle. In the rabbit, on the contrary to the rat, activity of the globular forms of AChE in a fast muscle increased after low-frequency stimulation. The results demonstrate that specific neural activation patterns regulate AChE activity in muscles. Great differences, however, exist among different mammalian species in regard to muscle AChE regulation. J. Neurosci. Res. 47:49–57, 1997. © 1997 Wiley-Liss, Inc.     

Stimulation of denervated rat soleus muscle with fast and slow activity patterns induces different expression of acetylcholinesterase molecular forms

The relative amount and distribution of acetylcholinesterase (AChE) molecular forms were studied in slow soleus and (less extensively) in fast extensor digitorum longus (EDL) muscles of the rat before and after denervation and direct stimulation. Normal EDL muscles showed higher total and specific AChE activity than normal soleus muscles and contained essentially three different molecular AChE forms (G1, G4, and A12) as opposed to six forms (G1, G2, G4, A4, A8, and A12) in the soleus. Denervation reduced AChE activity in both muscles. In the soleus direct stimulation starting 2 to 3 weeks after denervation increased the specific AChE activity markedly. The increase started 12 to 24 hr after the onset of stimulation, reached 3 to 5 times normal values after 2 to 7 days, and then declined gradually toward normal values over the next 2 weeks. Furthermore, the effect on the different molecular forms depended strongly on the stimulus pattern. Thus, intermittent 100 Hz stimulation (fast pattern) induced essentially the three forms typical of the normal EDL, whereas continuous 10 Hz stimulation induced the six forms characteristic of normal soleus muscles but with some differences in their relative proportions. In the EDL, 2 days of continuous 10 Hz stimulation (the only duration and pattern examined) failed to induce a similar increase in AChE activity.     

Effects of hindlimb unloading on neuromuscular development of neonatal rats

We hypothesized that hindlimb suspension unloading of 8-day-old neonatal rats would disrupt the normal development of muscle fiber types and the motor innervation of the antigravity (weightbearing) soleus muscles but not extensor digitorum longus (EDL) muscles. Five rats were suspended 4.5 h and returned 1.5 h to the dam for nursing on a 24 h cycle for 9 days. To control for isolation from the dam, the remaining five littermates were removed on the same schedule but not suspended. Another litter of 10 rats housed in the same room provided a vivarium control. Fibers were typed by myofibrillar ATPase histochemistry and immunostaining for embryonic, slow, fast IIA and fast IIB isomyosins. The percentage of multiple innervation and the complexity of singly-innervated motor terminal endings were assessed in silver/cholinesterase stained sections. Unique to the soleus, unloading accelerated production of fast IIA myosin, delayed expression of slow myosin and retarded increases in standardized muscle weight and fiber size. Loss of multiple innervation was not delayed. However, fewer than normal motor nerve endings achieved complexity. Suspended rats continued unloaded hindlimb movements. These findings suggest that motor neurons resolve multiple innervation through nerve impulse activity, whereas the postsynaptic element (muscle fiber) controls endplate size, which regulates motor terminal arborization. Unexpectedly, in the EDL of unloaded rats, transition from embryonic to fast myosin expression was retarded. Suspension-related foot drop, which stretches and chronically loads EDL, may have prevented fast fiber differentiation. These results demonstrate that neuromuscular development of both weightbearing and non-weightbearing muscles in rats is dependent upon and modulated by hindlimb loading.     

Comparison of Thyroid Function between Responders and Nonresponders to Thyroid Hormone Supplementation in Depression

Abstract: The serum levels of thyroxine (T₄), 3,5,3'-triiodothyronine (T₃) and 3,3',5'-triiodothyronine (reverse T₃, _rT₃) were examined from 8 depressed patients who did not maximally benefit from conventional antidepressant therapy. Four of the 8 depressed patients showed significant clinical improvement after thyroid hormone was added to their ongoing antidepressant drugs. The T₄ and _rT₃ levels prior to thyroid hormone supplementation were significantly lower in responders than in nonresponders, although within the normal range. Furthermore, all of the patients who had both their _rT₃ levels less than 200 pg/ml and T₄ levels less than 7 μ/dl responded to the thyroid hormone treatment. These data suggest that the lower T₄ and _rT₃ levels can predict the treatment response to the thyroid hormone supplementation in depressed patients.     

Regulation by exercise of the pool of G4 acetylcholinesterase characterizing fast muscles: opposite effect of running training in antagonist muscles

Fast muscles of rodents characteristically differ from their slow-twitch counterparts by exhibiting high levels of G4, i.e., the tetrameric acetylcholinesterase (AChE) molecular form. Converging evidence suggests that this additional G4 pool is specifically regulated by the type of activity actually performed by the muscle. This hypothesis was tested by studying the effect of a chronic increase in neuromuscular activity on the AChE content and distribution of molecular forms of functionally antagonist rat hindlimb muscles. They included the fast ankle extensors gastrocnemius (GAST) and plantaris (PL), the fast ankle flexors tibialis anterior (TA) and extensor digitorum longus (EDL), as well as the slow-twitch soleus (SOL). Neuromuscular activity was enhanced by subjecting the rats to a 12-week training program consisting of repeated sessions of prolonged endurance running on a rodent treadmill. This exercise regimen preferentially affected the G4 pool characterizing fast muscles which exhibited marked and opposite changes according to the functional role of the muscles. The amount of G4 was increased by more than 50% in the ankle extensors GAST and PL, which play a dynamic role, and reduced by about 40% in the ankle flexors TA and EDL, which exhibit a predominant tonic activity during running. The asymmetric forms A12 and A8 were slightly elevated in the fast muscles. In the case of the slow-twitch SOL, running training resulted in a small, nonspecific decrease in AChE content which affected most of the molecular forms. These data indicate that the size of the G4 pool characteristic of fast muscles depends on the type, dynamic or tonic, of activity actually performed. The present results support the conclusion that this G4 pool fulfills a specific and essential function, distinct from that of A12.     

Effect of resistance training on neuromuscular junctions of young and aged muscles featuring different recruitment patterns

To examine the effects of aging on neuromuscular adaptations to resistance training (i.e., weight lifting), young (9 months of age) and aged (20 months of age) male rats either participated in a 7‐week ladder climbing protocol with additional weight attached to their tails or served as controls (n = 10/group). At the conclusion, rats were euthanized and hindlimb muscles were quickly removed and frozen for later analysis. Longitudinal sections of the soleus and plantaris muscles were collected, and pre‐ and postsynaptic features of neuromuscular junctions (NMJs) were visualized with immunofluorescence staining procedures. Cross‐sections of the same muscles were histochemically stained to determine myofiber profiles (fiber type and size). Statistical analysis was by two‐way ANOVA (main effects of age and treatment) with significance set at P ≤ 0.05. Results revealed that training‐induced remodeling of NMJs was evident only at the postsynaptic endplate region of soleus fast‐twitch myofibers. In contrast, aging was associated with pre‐ and postsynaptic remodeling in fast‐ and slow‐twitch myofibers of the plantaris. Although both the soleus and the plantaris muscles failed to display either training or aging‐related alterations in myofiber size, aged plantaris muscles exhibited an increased expression of type I (slow‐twitch) myofibers in conjunction with a reduced percentage of type II (fast‐twitch) myofibers, suggesting early stages of sarcopenia. These data demonstrate the high degree of specificity of synaptic modifications made in response to exercise and aging and that the sparsely recruited plantaris is more vulnerable to the effects of aging than the more frequently recruited soleus muscle. © 2014 Wiley Periodicals, Inc.     

Acetylcholinesterase activity in motor nerve fibres in correlation to muscle fibre types in rat

Rat muscle nerves were examined histochemically for their activity of acetylcholinesterase (AChE). The corresponding muscles were stained for myofibrillar ATPase and for NADH diaphorase. The nerves to the extensor digitorum longus (EDL) muscle and to the medial head of the gastrocnemius (MG) muscle consist of a motor axons of high AChE activity. Both muscles are characterized by the prevalence of type II muscle fibres. On the other hand, the soleus muscle and the quandratus femoris muscle, both mainly composed of type I muscle fibres, are innervated by a motor axons of low AChE activity. Since it is well established that EDL and MG are typical fast-twitch muscles and that the soleus, and probably also the auadratus femoris, is a typical slow-twitch muscle, it is suggested that, in rat, fast muscles are innervated by motor nerve fibres of high AChE activity and slow muscles are innervated by motor axons of low AChE activity.     

KINETICS OF CELL PROLIFERATION IN THE POSTNATAL RAT DENTATE GYRUS

Cell cycle parameters in the dentate gyrus of the postnatal rat hippocampus were measured, using the method of percentage labelled mitoses. In animals aged 1, 6 and 12 days, cell cycle times range from 15-1 to 17-7 h, S phase duration from 10-1 to 11-7 h, length of the G₂ phase from 2–5 to 3-3 h and G₁ from 1-1 to 2–4 h. These figures are similar to those obtained for other precursor cell populations in the brain. On the day after birth the labelling index in the dentate hilus is 4–5% and estimated turnover time 220 h, while on day 6 the labelling index is 3–7% and estimated turnover time 310 h. It is calculated that precursor cells outnumber granule cells by at least 3:1 at birth and 2:1 by day 6.