Morphological and membrane properties of rat magnocellular basal forebrain neurons maintained in culture

Morphological and electrophysiological characteristics of magnocellular neurons from basal forebrain nuclei of postnatal rats (11-14 days old) were examined in dissociated cell culture. Neurons were maintained in culture for periods of 5-27 days, and 95% of magnocellular (>23 micron diam) neurons stained positive with acetylcholinesterase histochemistry. With the use of phase contrast microscopy, four morphological subtypes of magnocellular neurons could be distinguished according to the shape of their soma and pattern of dendritic branching. Corresponding passive and active membrane properties were investigated with the use of whole cell configuration of the patch-clamp technique. Neurons of all cell types displayed a prominent (6-39 mV; 6.7-50 ms duration) spike afterdepolarization (ADP), which in some cells reached firing threshold. The ADP was voltage dependent, increasing in amplitude and decreasing in duration with membrane hyperpolarization with an apparent reversal potential of -59 +/- 2.3 (SE) mV. Elevating [Ca2+]o (2.5-5.0 mM) or prolonging spike repolarization with 10 mM tetraethylammonium (TEA) or 1 mM 4-aminopyridine (4-AP), potentiated the ADP while it was inhibited by reducing [Ca2+]o (2.5-1 mM) or superfusion with Cd2+ (100 microM). The ADP was selectively inhibited by amiloride (0.1-0.3 mM or Ni2+ 10 microM) but unaffected by nifedipine (3 microM), omega-conotoxin GVIA (100 nM) or omega-agatoxin IVA (200 nM), indicating that Ca2+ entry was through T-type Ca2+ channels. After inhibition of the ADP with amiloride (300 microM), depolarization to less than -65 mV revealed a spike afterhyperpolarization (AHP) with both fast and slow components that could be inhibited by 4-AP (1 mM) and Cd2+ (100 microM), respectively. In all cell types, current-voltage relationships exhibited inward rectification at hyperpolarized potentials >/=EK (approximately -90 mV). Application of Cs+ (0.1-1 mM) or Ba2+ (1-10 microM) selectively inhibited inward rectification but had no effect on resting potential or cell excitability. At higher concentrations, Ba2+ (>10 microM) also inhibited an outward current tonically active at resting potential (VH -70 mV), which under current-clamp conditions resulted in small membrane depolarization (3-10 mV) and an increase in cell excitability. Depolarizing voltage commands from prepulse potential of -90 mV (VH -70 mV) in the presence of tetrodotoxin (0.5 microM) and Cd2+ (100 microM) to potentials between -40 and +40 mV cause voltage activation of both transient A-type and sustained delayed rectifier-type outward currents, which could be selectively inhibited by 4-AP (0.3-3 mM) and TEA (1-3 mM), respectively. These results show that, although acetylcholinesterase-positive magnocellular basal forebrain neurons exhibit considerable morphological heterogeneity, they have very similar and characteristic electrophysiological properties.     

Spontaneous electromyographic activity in adult rat soleus muscle

Single-motor-unit and gross electromyograms (EMG) were recorded from the soleus muscle in six unrestrained rats. The median firing frequencies of nine motor units were in the 16-25 Hz range, in agreement with previous studies. One additional motor unit had a median firing frequency of 47 Hz. This unit and one of the lower-frequency units regularly fired doublets. Motor-unit firing frequency was well correlated to whole-muscle EMG during locomotion. Integrated rectified gross EMG revealed periods of continuous modulation, phasic high-amplitude events, and tonic low-amplitude segments. The tonic segments typically were caused by a small number of motor units firing at stable high frequencies (20-30 Hz) for extended periods of time without detectable activity in other units. This long-lasting firing in single motor units typically was initiated by transient mass activity, which recruited many units. However, only one or a few units continued firing at a stable high frequency. The tonic firing terminated spontaneously or in conjunction with an episode of mass activity. Different units were active in different tonic segments. Thus there was an apparent dissociation between activity in different single motor units and consequently between single-motor-unit activity and whole-muscle EMG. It is proposed that the maintained tonic motor-unit activity is caused by intrinsic motoneuron properties in the form of depolarizing plateau potentials.     

Free radical involvement in doxorubicin-induced electrophysiological alterations in rat papillary muscle fibres

A simple methodology for the manufacture and calibration of polyacrylamide gel (PAG) for magnetic resonance imaging (MRI) radiation dosimetry is presented to enable individuals to undertake such work in a routine clinical environment. Samples of PAG were irradiated using a linear accelerator and imaged using a 0.5 T (22 MHz) Philips Gyroscan MRI scanner. The mean spin-lattice relaxation rate was measured using a 'turbo-mixed' sequence, consisting of a series of 90 degrees pulses, each followed by acquisition of a train of spin echoes. The mean sensitivity for five different batches of PAG in the range up to 10 Gy was calculated to be 0.0285 s-1 Gy-1 for the mean spin-lattice relaxation rate with a percentage standard deviation of 1.25%. The overall reproducibility between batches was calculated to be 2.69%. This methodology, which introduces the novel use of pre-filled nitrogen vials for calibration, has been used to develop techniques for filling anatomically shaped anthropomorphic phantoms.     

Calcium homeostasis in rat septal neurons in tissue culture

Septal neurons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca2+ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca2+]i) in the neurons was low (50-100 nM). Depolarization of the neurons with 50 mM K+ resulted in rapid elevation of [Ca2+]i to 500-1,000 nM showing recovery to baseline [Ca2+]i over several minutes. The increases in [Ca2+]i caused by K+ depolarization were completely abolished by the removal of extracellular Ca2+, and were reduced by approximately 80% by the 'L-type' Ca2+ channel blocker, nimodipine (1 microM). [Ca2+]i was also increased by the excitatory amino acid L-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNQX and DNQX. In the absence of extracellular Mg2+, large fluctuations in [Ca2+]i were observed that were blocked by removal of extracellular Ca2+, by tetrodotoxin (TTX), or by antagonists of N-methyl D-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg2+ and TTX, NMDA caused dose-dependent increases in [Ca2+]i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca2+]i in the absence of extracellular Ca2+, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca2+ stores. Thapsigargin (2 microM) had little effect on [Ca2+]i, or on the recovery from K+ depolarization. Removal of extracellular Na+ had little effect on basal [Ca2+]i or on responses to high K+, suggesting that Na+/Ca2+ exchange mechanisms do not play a significant role in the short-term control of [Ca2+]i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca2+]i; however, [Ca2+]i recovered as normal from high K+ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca2+]i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca2+]i associated with action potential activity was measured. The amplitude of the [Ca2+]i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca2+]i from the modest Ca2+ loads imposed on the neuron by action potential trains follows a simple exponential decay (tau = 3-5 s).     

Force and myosin content variation in isolated intact single muscle fibres from Rana temporaria

We studied the relation between force normalized by dry mass per unit length and the myosin fraction of muscle dry mass. The two tibialis anterior muscles were dissected from 12 frogs (Rana temporaria). Then, from one muscle, two single fast-twitch fibres were isolated. Each fibre was mounted isometrically in Ringer's solution, and electrically stimulated using a standardized protocol. Peak force production, normalized by the fibre's dry mass per unit length, varied by a factor of 1.4. Little variation in normalized force was measured between fibres from the same animal, whereas between animals a significant difference was found (P<0.05). The contralateral muscle was used to determine the myosin fraction of the dry mass. The relationship between the fraction myosin of the dry mass and force normalized by dry mass per unit length showed a high correlation (r = 0.81; n = 12). From this we conclude that variation in normalized tetanic force is determined greatly (65%) by variations in myosin content.     

Sensory terminals on extrafusal muscle fibres in myotendinous regions of developing rat muscles

Axon terminals were observed to form neuromuscular contacts with extrafusal muscle fibres in myotendinous regions of developing rat muscles up to 5 days after birth. These neuromuscular contacts are found in fascicles of muscle fibres connected with differentiating Golgi tendon organs. Axon terminals establishing these contacts are obviously sensory, since they do not degenerate after de-efferentation performed in neonatal rats. The terminals contain mainly clear and dense core vesicles and form neuromuscular connections resembling developing motor endplates, with a cleft about 60 nm wide and basal lamina interposed between the axolemma and the sarcolemma. Each terminal, however, also forms a close contact in a restricted region where the basal lamina is missing; there the cleft is reduced to 20 nm and the axolemmal and sarcolemmal membranes are linked by desmosome-like attachment plaques. After the fifth postnatal day, axon terminals become detached from muscle fibres and are only found among collagen bundles of the tendon organ. The functional significance of these temporary neuromuscular contacts is not clear.     

Electron microscopy of postmortem autolysis of rat muscle tissue

To define the progression of ultrastructural changes in normal muscle at post mortem, rat gastrocnemius muscles were studied at various times after storage at +4 degrees C and +22 degrees C. Degeneration of the I-zone (discoid necrosis) and membranous bodies were found to be similar to that seen in muscle diseases, and lamellar formations were seen in mitochondria. At +4 degrees C there was contraction of the sarcomere which vanished in 12 h and inter-filamentous oedema appeared. Z-line degeneration was seen at 24 h and at 4 days all Z-lines had disappeared, and the H-zone showed darkening. In the same samples collapse or ruptures of the I band were seen. At 8 days the H-zones and M-lines were still discernible. In the early stages the mitochondria showed swelling and loss of matrix granules, while later they showed broken cristae and outer membranes, and flocculent densities. At 4 days rearrangement of the cristal material into long pentalaminar "needles" was seen in a few mitochondria. At 4 and 8 days membranous bodies were seen and the T-system and sarcoplasmic reticulum showed ruptures and disintegration into vesicles. The nucleus showed increasing condensation of chromatin at the periphery and clearing of the center. Polysomes and glycogen were reduced at 2 h, and has practically vanished at 24 h. At 22 degrees C the changes were the same but appeared about 4 times as quickly as at +4 degrees C.     

Force production in mechanically isolated cardiac myocytes from human ventricular muscle tissue

OBJECTIVE: The expression of contractile isoforms changes during various pathological conditions but little is known about the consequences of these changes for the mechanical properties in human ventricular muscle. We investigated the feasibility of simultaneous determination of protein composition and isometric force development in single cardiac myocytes from human ventricular muscle tissue obtained from small biopsies taken during open heart surgery. METHODS: Small biopsies of about 3 mg wet weight were taken during open heart surgery from patients with aortic valve stenosis. These biopsies were divided in two parts. One part (approximately 2 mg) was used for mechanical isolation of single myocytes and subsequent force measurement while the remaining part was used, in aliquots of 1 microgram dry weight, for protein analysis by polyacrylamide gel electrophoresis. The myocytes were attached with silicon glue to a sensitive force transducer and a piezoelectric motor, mounted on an inverted microscope and permeabilized by means of Triton X-100. Force development was studied at various free calcium concentrations. RESULTS: From all biopsies, myocytes could be obtained and the composition of contractile proteins could be determined. The average isometric force (+/- s.e.m.) at saturating calcium concentration obtained on 20 myocytes from 5 patients amounted to 51 +/- 8 kN/m2. Force was half maximal at a calcium concentration of 2.47 +/- 0.10 microM. CONCLUSION: These measurements indicate that it is possible to study the correlation between mechanical properties and protein composition in small biopsies from human ventricular muscle.     

Branched-chain amino acid oxidation by isolated rat tissue preparations

Branched-chain amino acid transaminase activity, branced-chain alpha-keto acid dehydrogenase activity, and leucine oxidation were measured in homogenates and slices of several rat tissues. Transaminase activity was highest in heart, while dehydrogenase activity was highest in liver. Leucine oxidation in isolated tissues may be limited by either transaminase or dehydrogenase activity depending upon the relative activities of these two enzymes in the tissue. The results suggest that, as the load of branched-chain amino acids increases, the liver may become an increasingly important site for the degradation of branched-chain alpha-keto acids.     

Characterization of prostaglandin production in tissue culture of rat renal medullary cells

The purpose of our studies was to examine the role of the nervous system in arrhythmias produced by digitalis overdose and coronary artery occlusion in the cat. This was done by observing the effect of these arrhythmogenic procedures on cardiac efferent neural activity and then determining whether any observed alteration in neural activity contributed to the cardiac rhythm disturbances evoked by digitalis and coronary artery occlusion. Our data indicate that both procedures used to evoke arrhythmias activate each division of the autonomic nervous system. Activation of the sympathetic nervous system resulted in a deleterious effect on cardiac rhythm whereas activation of the parasympathetic nervous system, in general, resulted in a beneficial effect on cardiac rhythm. With coronary occlusion, the role exerted by the nervous system depended on the anatomic location of the involved myocardium. Studies directed at elucidating the mechanisms whereby the nervous system caused cardiac rhythm disturbances indicated that there may be an important difference between the antiarrhythmic efficacy of beta-adrenergic blockade and bilateral stellate ganglionectomy. The latter procedure proved to be a more effective way of removing deleterious sympathetic neural effects on the heart. In conclusion, our findings suggest that the development of new drugs for treating arrhythmias resulting from digitalis and coronary occlusion should be aimed at finding drugs that act to either depress central sympathetic outflow or enhance parasympathetic effects on the ventricle.     

Oxidative stress and release of tissue plasminogen activator in isolated rat hearts

To evaluate the potential of tissue plasminogen activator (t-PA) as a marker of endothelial activation or injury, the dose-response relationship between reactive oxygen intermediates and t-PA release was investigated in isolated rat hearts. After stabilization the hearts were perfused for 10 minutes with different concentrations of hydrogen peroxide (H2O2) (0 (control perfusion), 20, 40, 80, 120, 160, or 200 microM) (n = 8 hearts/group), followed by 30 minutes recovery. Higher concentrations than 80 microM induced cardiac dysfunction and a dose-dependent release of lactate dehydrogenase, indicating myocyte injury. H2O2-concentrations of 80 microM and more caused a significant, but temporary t-PA release. Peak t-PA release occurred more rapidly with higher concentrations, but otherwise there was no difference dependent on the H2O2-dose. The effects of H2O2 (120 or 200 microM) on t-PA release were also compared to the effects of bradykinin. Both were given for 10 minutes as above, and the procedure was repeated after 10 minutes recovery. Bradykinin (50 or 500 nM) released t-PA with the same magnitude, but with peak values occurring earlier than t-PA release induced by H2O2. Bradykinin, but not H2O2, induced t-PA release during the second exposure, suggesting different mechanisms of release. In conclusion: Perfusion with H2O2 leads to a dose-dependent myocardial injury in isolated rat hearts. H2O2 also causes an acute t-PA release without dose-dependency, suggesting an all or nothing response of the endothelium. t-PA may be used as an indicator of, but cannot quantify endothelial activation or injury.     

The use of Zn-desferrioxamine for radioprotection in mice, tissue culture, and isolated DNA

We previously reported that nitric oxide (NO) production in the unipolar brush (UB) cells is involved in vestibular compensation [T. Kitahara, N. Takeda, P.C. Emson, T. Kubo, H. Kiyama, Changes in nitric oxide synthase-like immunoreactivities in unipolar brush cells in the rat cerebellar flocculus after unilateral labyrinthectomy, Brain Res. 765 (1997) 1-6]. To further elucidate the role of NO-mediated signaling in flocculus after unilateral labyrinthectomy (UL), we examined UL-induced Fos expression, a marker of neural activity, in vestibular brainstem with continuous floccular infusions of Nomega-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthase (NOS). After UL with floccular l-NAME infusions, Fos expression appeared in bilateral medial vestibular (MVe) and prepositus hypoglossal (PrH) nuclei. After UL with floccular saline infusions, however, Fos expression was observed only in the ipsi-MVe and contra-PrH. Furthermore, it has been revealed that UL with l-NAME infusions caused more severe vestibulo-ocular disturbances than UL with saline infusions at the initial stage [Kitahara et al. Brain Res. 765 (1997) 1-6]. Therefore, it is suggested that UL with floccular l-NAME infusions activates the contra-MVe and ipsi-PrH neurons and causes more severe imbalance between intervestibular nuclear activities at the initial stage. NO-mediated signaling in flocculus could be a possible driving force of the flocculus-mediated inhibition on the contra-MVe and ipsi-PrH at the initial stage of vestibular compensation.     

Electron-microscopic radioautography of DNA-synthesizing cells of growing rat smooth muscle tissue

DNA-synthesizing cells of rat stomach muscle tissue following 50% resection of the fundal part, identical cells of the vena cava after disturbance of the blood out flow, and of the appendix on contraction of the ascending part of the large intestine were studied by electron microscopic autoradiography. DNA synthesis in the differentiated myocyte nuclei of the stomach muscle tissue, and of the "activated" myocytes of the stomach and the vein was observed. There were signs of asynchronous DNA synthesis in the nuclei of some smooth muscle cells and fibroblasts.     

Neurokinin A in rat renal afferent neurons and in nerve fibres within smooth muscle and epithelium of rat and guinea-pig renal pelvis

Neurokinin A-like immunoreactivity of dorsal root ganglion neurons innervating the kidney were studied with retrograde tracing of FluoroGold dye applied to the cut renal nerves. The proportions and sizes of renal afferent neurons with neurokinin A-like immunoreactivity were quantified in T9-L2 dorsal root ganglia from five rats. Of 240 renal afferent neuronal somata examined, 26 +/- 3% (S.E.M.) showed neurokinin A-like immunoreactivity. Compared with the overall size distribution of renal afferent neurons, those staining for neurokinin A were mostly small-sized neurons with a few medium-sized neurons. All somata with neurokinin A-like immunoreactivity were neurofilament-poor as judged by labelling with an anti-neurofilament antibody, RT97, and it is therefore likely that they had unmyelinated fibres. To examine the sites to which the renal afferent fibres with neurokinin A might project, sections of rat and guinea-pig kidney and upper ureter were examined. Fibres with neurokinin A-like immunoreactivity were found beneath and within the transitional epithelium lining the inner surface of the pelvis, and within the smooth muscle layer beneath the transitional epithelium. Epithelial innervation was found only in regions with underlying smooth muscle and loose connective tissue, and not in sites where the epithelium was closely applied to the renal parenchyma. The network of fibres was most dense towards the pelvo-uretic junction. Fibres with neurokinin A-like immunoreactivity were not seen beneath or within the cuboidal/columnar epithelium covering the papilla within the renal pelvis. Furthermore, only very few fibres with neurokinin A were observed penetrating the transitional epithelium of the upper ureter in both rat and guinea-pig. The distribution of fibres labelled with antibodies to substance P and calcitonin gene-related peptide in the renal pelvis was similar to that for fibres with neurokinin A-like immuno-reactivity, although a few fibres penetrated further into the fornices than fibres with neurokinin-A-like immunoreactivity. Thus, many afferent fibres in the renal pelvis may contain neurokinin A as well as substance P and calcitonin gene-related peptide. These fibres may be the source of the neurokinin A, substance P and calcitonin gene-related peptide which can be released by topical capsaicin treatment. In addition they may be the mechano- and chemo-receptive fibres in the renal pelvis that are known to play important roles in renal haemodynamics. The intra-epithelial position of some of these fibres in the epithelial layer suggests a possible chemosensory or osmosensory role.     

Transmitter-induced calcium responses differ in astrocytes acutely isolated from rat brain and in culture

Glial fibrillary acid protein (GFAP)-positive astrocytes isolated from the cerebral cortices of 3-10-day-old rats frequently showed increased intracellular Ca2+ concentration responses to L-glutamate and glutamate analogues. However, few of the acutely isolated cells responded to ATP, and no such cells responded to serotonin [5-hydroxytryptamine (5-HT)]. The same cell that failed to respond to ATP or 5-HT often responded to glutamate. Culturing acutely isolated cells in media containing horse serum decreased Ca2+ responses to glutamate but increased the responses to ATP and induced responses to 5-HT. In primary cultures prepared from the cerebral cortices of 1-day-old rats and cultured in horse serum, fewer of the cells responded to glutamate, but almost all cells responded to ATP and 5-HT. The lack of or limited response to, 5-HT or ATP in the acutely isolated cells seems unlikely to be due to selective damage to the respective receptors because acutely isolated GFAP-negative cells showed responses to ATP, several different proteases and mechanical dissociation yielded cells that also responded to glutamate but not to ATP, and exposure of primary cultures to papain did not abolish Ca2+ responses to several transmitters. The responses of the acutely isolated cells to glutamate but limited or lack of responses to ATP and 5-HT also correspond to what has been seen so far for astrocytes in situ. Thus, the present studies provide direct evidence that some of the receptors seen in primary astrocyte cultures may reflect a response to culture conditions and that, in the context of the relevant information so far available, acutely isolated astrocytes seem to reflect better the in vivo state.