大鼠脑梗塞灶周围区GAP-43免疫组化反应的观察

目的 探查脑梗塞灶周围区GAP-43表达的结构和时间.方法 运用电凝阻断大脑中动脉脑局部缺血模型和免疫组化(ABC)染色方法,对38只SD大鼠进行了研究.结果 GAP-43阳性反应发生于脑梗塞灶周围区的神经元的胞体和突起.其表达高峰在脑梗塞3～7d,随后逐渐减弱.神经元突起的阳性反应显示不同的形式和时相,其反应部位邻近梗塞区,在脑缺血2～3w,其显示强反应,没有发现胶质细胞的阳性反应.结论 局灶性脑缺血诱导GA-43蛋白质在梗塞灶周围区神经元表达,提示缺血区周边皮质神经元有生长和修复反应倾向.     

Intracellular calcium and hormone release from nerve endings of the neurohypophysis in the presence of opioid agonists and antagonists

Summary Rat neural lobes and isolated nerve terminals from the neurohypophysis were stimulated in the presence of different opioid agonists and antagonists. The secretion of arginine vasopressin and oxytocin and rise in cytoplasmic calcium induced by depolarization were analyzed by radioimmunoassay and the fluorescent probe fura-2, respectively. The kappa-agonists dynorphin A_{1 -13} and dynorphin A_{1 -8} did not affect electrically evoked release of vasopressin, although oxytocin release was slightly reduced. U-50 488, a relatively specific kappa-receptor agonist, had no effect on the amount of vasopressin or oxytocin secreted, although it significantly reduced K⁺-evoked changes in [Ca²⁺]_i in isolated nerve endings. Two kappa-receptor antagonists, MR 2266 and diprenorphin, alone had no effect on vasopressin and oxytocin secretion from isolated nerve endings depolarized with potassium. Opioid agonists less selective for the kappa receptors, etorphin and ethylketocyclazocin, were found to inhibit the release of both vasopressin and oxytocin significantly. Naloxone, a nonselective opiate receptor antagonist, alone had no effect on vasopressin release but potentiated the electrically evoked release of oxytocin. Naloxone also could overcome the inhibitory effect of etorphin on oxytocin and vasopressin release observed after electrical stimulation of the neural lobe. A number of inconsistencies therefore exist between the effects of opioid agonists and antagonists on neuropeptide release and on the evoked changes in [Ca²⁺]_i. In view of these inconsistencies and the high concentrations of opioid agonists and antagonists necessary to modify release, we conclude that it is doubtful that opioid molecules have a physiological role in controlling neurohypophysial secretion.     

The disruption of myofibre structures in rat skeletal muscle after forced lengthening contractions

 Specific antibodies against structural proteins (actin, desmin, dystrophin, fibronectin) of muscle fibres were used to study the effect of forced lengthening contractions on muscle microarchitecture. Tibialis anterior (TA) muscle of male Wistar rats were subjected to 240 forced lengthening contractions. At consecutive time points (0, and 6 h, 2, 4, and 7 days) after stimulation, the TA muscle was excised for biochemical and histological assays. β-Glucuronidase activity, a quantitative indicator of muscle damage, showed increased values 2–7 days after the lengthening, peaking on day 4 (11.7-fold increase). A typical course of histopathological changes (myofibre swelling, necrosis and regeneration) was observed. In immunohistochemistry, the earliest abnormality observed was discontinuous dystrophin staining in some swollen fibres immediately after commencement of exercise, while at the same time no alterations occurred in the staining of the other antibodies studied. Six hours later, all the swollen fibres were uniformly desmin as well as dystrophin negative. The great majority, but not all, of the swollen fibres showed disorganized actin staining and intramyocellular localization of fibronectin. The early phase disruption of myofibre structures as measured in this study provides evidence of their central role following damage in skeletal muscle. These results suggest that the sequence of structural changes in the route to muscle fibre necrosis in injury induced by forced lengthening contraction originates in the disruption of the plasma membrane and the intermediate filament, which leads to disturbances in the myofibrillar system. Received: 26 January 1998 / Received after revision: 12 May 1998 / Accepted: 14 May 1998     

Changes in trkA expression in the dorsal root ganglion after peripheral nerve injury

Most of the biological effects of nerve growth factor (NGF) are mediated by TrkA, the high affinity receptor for NGF. Previous studies have shown that NGF levels in the dorsal root ganglia (DRG) fluctuate following a peripheral nerve injury. The present study examined changes of TrkA immunoreactivity and trkA mRNA expression in the DRG after segmental nerve ligation. In the normal L5 DRG of the rat, there were, on average, 4700 TrkA-immunoreactive (TrkA-IR) neurons, representing 42% of the total neuronal population. Following L5 spinal nerve ligation, the number of TrkA-IR neurons in the L5 DRG slowly declined, reducing by 25% at 1 week and 35% at 3 weeks postoperation (PO). In contrast, trkA mRNA in these ganglia showed a significant decrease from 3 days to 3 weeks PO and was followed by a full recovery at 2 months PO. The early decrease of trkA mRNA is likely due to deprivation of target-derived NGF, which is caused by nerve ligation, and the recovery might be because substitute sources of NGF become available. Despite the decline in trkA mRNA in the ganglion, 3000 injured DRG neurons sustain TrkA immunoreactivity, suggesting that exogenous NGF can still influence these TrkA expressing neurons, even though they are isolated from the periphery. Accordingly, the effects of endogenous NGF should be as well manifested by local administration of NGF to the ganglion as to the stump of the damaged nerve. Received: 20 October 1998 / Accepted: 10 February 1999     

Expression of two neuronal markers, growth-associated protein 43 and neuron-specific enolase, in rat glial cells

 Recent studies have revealed that proteins such as growth-associated protein 43 (GAP-43) and neuron-specific enolase (NSE), believed for many years to be expressed exclusively in neurons, are also present in glial cells under some circumstances. Here we present an overview of these observations. GAP-43 is expressed both in vitro and in vivo transiently in immature rat oligodendroglial cells of the central nervous system, in Schwann cell precursors, and in non-myelin-forming Schwann cells of the peripheral nervous system. GAP-43 mRNA is also present in oligodendroglial cells and Schwann cells, indicating that GAP-43 is synthesized in these cells. GAP-43 is also expressed in type 2 astrocytes (stellate-shaped astrocytes) and in some reactive astrocytes but not in type 1 astrocytes (flat protoplasmic astrocytes). These results suggest that GAP-43 plays a more general role in neural plasticity during development of the central and peripheral nervous systems. NSE enzymatic activity and protein and mRNA have been detected in rat cultured oligodendrocytes at levels comparable to those of cultured neurons. NSE expression increases during the differentiation of oligodendrocyte precursors into oligodendrocytes. In vivo, NSE protein is expressed in differentiating oligodendrocytes and is repressed in fully mature adult cells. The upregulation of NSE in differentiating oligodendrocytes coincides with the formation of large amounts of membrane structures and of protoplasmic processes. Similarly, NSE becomes detectable in glial neoplasms and reactive glial cells at the time when these cells undergo morphological changes. The expression of the glycolytic isozyme NSE in these cells, which do not normally contain it, could reflect a response to higher energy demands. This expression may also be related to the neurotrophic and neuroprotective properties demonstrated for this enolase isoform. NSE activity and protein and mRNA have also been found in cultured rat type 1-like astrocytes but at much lower levels than in neurons and oligodendrocytes. Thus GAP-43 and NSE should be used with caution as neuron-specific markers in studies of normal and pathological neural development. Received: 27 January 1997 / Accepted: 9 May 1997     

Subcellular localization of low-affinity nerve growth factor receptor-immunoreactive protein in adult rat purkinje cells following traumatic injury

Cerebellar Purkinje cells in the rat express low-affinity nerve growth factor receptor (p75 NGFR) antigen during development, but rarely in normal adult animals. In striking contrast, re-expression of p75 NGFR-immunoreactive protein was reported by light microscopy immunocytochemistry in adult rat Purkinje cells as early as 1 day after traumatic axotomy. Characteristically, varicose axons through the infraganglionic zone were also stained. To date, however, there is no information on the subcellular location of the antigenic re-expression. To address this, a pre-embedding immunocytochemical ultrastructural study using affinity-purified monoclonal 192-IgG was carried out after an experimentally induced traumatic lesion of the rat cerebellum. At the electron microscopic level, immunostaining was intense in Purkinje cells. In these cells, the immunoreactivity was always associated with the internal face of the membranes of the rough endoplasmic reticulum, Golgi apparatus and nuclear envelope. Patches of immunoreactivity were also associated with the outer surface of the plasma membrane of the cell body, dendritic processes and axons. It is noteworthy that receptor immunoreactivity was detected in recurrent collaterals of Purkinje cell axons forming symmetric synaptic contacts with the cell body and dendrites of immunonegative local circuit neurons. Results of this study show that injury-induced re-expression of p75 NGFR antigen is restricted to Purkinje cells. Also, the relative importance of the contribution of the local circuit neurons to the production of neurotrophic substances after trauma is suggested. Received: 28 October 1996 / Accepted: 1 September 1997     

Low-frequency electro-acupuncture reduces the nociceptive response and the pain mediator enhancement induced by nerve growth factor

A number of studies have shown that the potential clinical benefits of nerve growth factor (NGF) administration are limited by its hyperalgesic side effects. The ancient therapeutic technique of acupuncture and its modern derivate electro-acupuncture (EA) have been proven effective in reducing hyperalgesia as well as nociceptive and neuropathic pain in several pathological conditions. The present study addresses the question of whether EA can influence the hyperalgesia induced by NGF administration. We treated adult healthy rats with repeated injections of murine NGF and/or low-frequency electro-acupuncture. We found that EA was able to counteract the NGF-induced hyperalgesic response when assessed by a hot plate test. Moreover, EA counteracted the NGF-driven variation of substance P (SP) and transient receptor potential vanilloid type 1 (TRPV1) response in both hind-paw skin as well as the corresponding dorsal root ganglia (DRG). Our findings indicate that low-frequency EA could be useful as a supportive therapy to reduce NGF-induced side effects, such as hypersensitivity and hyperalgesia, when clinical treatment with NGF is necessary.     

Excitation of cutaneous afferent nerve endings in vitro by a combination of inflammatory mediators and conditioning effect of substance P

Summary A broad mixture of inflammatory mediators (inflammatory soup) was used to investigate the responsiveness of primary afferents from rat hairy skin in an in vitro skin-saphenous nerve preparation. In addition, a conditioning effect of the tachykinin substance P on chemosensitivity of nociceptors was examined. Inflammatory soup (IS) was made up in synthetic interstitial fluid from bradykinin, serotonin, histamin and prostaglandin E₂ (all 10^-5 M). In addition, the potassium and the hydrogen ion concentration (7 mM, pH 7.0) and the temperature (39.5°C) were elevated. The latter agents, in a control solution, did not excite nociceptors (n = 5). IS was repeatedly superfused over the receptive fields for 5 min at 10 min intervals; substance P (SP 10^-6 and 10^-5 M) was applied during the last 5 min of the interval and during the subsequent IS stimulation. IS excited more than 80% of the mechano-heat sensitive (polymodal) afferents with slowly conducting nerve fibres (n = 72), but none of the low-threshold mechanoreceptive slow and fast conducting units (n = 17). Slow conducting afferents with high mechanical threshold (n = 35) were weakly, and less frequently (<20%), driven by IS. A majority, but not all, of the responsive units showed tachyphylaxis upon repeated IS application. None, however, lost its responsiveness completely. Conditioning heat stimulation (32–46.5°C in 20 s) did not enhance the subsequent IS response, which may indicate that sensitizing substances normally released by a noxious heat stimulus were already contained in IS. No sensitization to mechanical (von Frey) or heat stimulation could be established in the period after the IS response had subsided and after the washout was completed, respectively. A short-lived sensitization may have been overlooked under these temporal restrictions. Conditioning SP in 10^-5 M but not in 10^-6 M concentration significantly increased the IS response of polymodal C fibres, by 58% on average (n = 14). SP did not excite the units. Comparing with previous data, we conclude that there is a significant synergism between inflammatory mediators, acting to induce more intense and more sustained discharge via many nociceptors than single mediators alone could achieve. Conditioning substance P can further enhance this algogenic action. Mechanisms of interaction and relative contributions of single substances remain to be elucidated.     

Adaptations of the β-adrenoceptor-adenylyl cyclase system in rat skeletal muscle to endurance physical training

 β-Adrenergic mechanisms may be important in the adaptation of skeletal muscle to endurance training. β-Adrenergic signal transduction was examined in the gastrocnemius muscle of rats submitted to a progressive, 12-week treadmill running program and compared with sedentary controls. β-Adrenoceptor density was significantly lower in exercised rats than in controls. The affinity constant for [¹²⁵I]-(-) iodocyanopindolol binding was not different among the various groups. Adenosine cyclic monophosphate formation was significantly decreased in trained animals when isoproterenol plus guanosine triphosphate or forskolin plus Mn²⁺ were used to stimulate adenylyl cyclase. Immunoblot analyses revealed that the amount of the α-subunit of stimulatory guanine nucleotide-binding protein (G_s,α), both the small and the large isoforms, also decreased with physical exercise. Thus, the present report shows that endurance training results in alterations in β-adrenergic receptor density, adenylyl cyclase activity and G_s protein level in rat gastrocnemius muscle. Received: 18 October 1996 / Received after revision: 11 March 1997 / Accepted: 20 June 1997     

实验性哮喘时神经生长因子对P物质的上调作用

目的：探讨实验性哮喘时神经生长因子（NGF）对P物质（SP）的调节机制。 方法： 应用放射免疫分析方法检测了NGF缺失时（鼻腔吸入NGF抗体）哮喘豚鼠下呼吸道和内脏感觉传入部位SP含量的变化。 结果： NGF缺失时哮喘豚鼠气管、支气管、肺、C7-T5段脊神经节及相应节段脊髓后角、结状神经节和孤束核区SP含量明显低于哮喘组和对照组（P<0.01）。 结论： 实验性哮喘时NGF可上调下呼吸道和感觉神经节等内脏感觉传入部位的P物质水平，两者共同参与支气管哮喘的发病过程。     

Development of the cyto- and chemoarchitectural organization of the rat nucleus of the solitary tract

The nucleus of the solitary tract (NST) is the major visceral sensory nucleus in the brainstem. The development of the rat nucleus of the solitary tract was followed during late prenatal and early postnatal life in order to determine when subnuclear organization and chemoarchitectural features develop. In Nissl-stained sections, the nucleus of the solitary tract becomes visible as a distinct cluster of cells by about E17. Between E17 and E19, a profound change in the Nissl-stained appearance of the nucleus occurred, so that by E19 all the subnuclei were discernible. Acetylcholinesterase activity in the developing NST showed an early period of rapid differentiation (E15 to E17), while by E19 the basic adult pattern of distribution of this enzyme had already been achieved. The subnuclei of the NST began to show clear differential staining for nicotinamide adenine dinucleotide phosphate diaphorase at about the same time as reactivity for that enzyme first appeared (E19). With respect to calbindin- and calretinin-immunoreactive neurons within the nucleus, many of the chemoarchitectural features associated with these two markers were obvious even by late fetal life. For example, in the central subnucleus, a strongly labelled, dense population of calbindin-immunoreactive neurons was present from E17; while in calretinin-immunoreacted material, this subnucleus was prominent because of its immunonegativity also from E17. Nevertheless, the total number of calbindin- and calretinin-immunoreactive neurons in the NST did not peak until late postnatal life. Tyrosine hydroxylase immunoreactive neurons were visible from E15, began differentiation by E17 and were distributed in a similar pattern to the adult from E19. Substance P immunoreactivity in the NST was also very similar to the adult pattern by E19. Many of these immunochemical and histochemical markers indicate a similar pattern of development, i.e. a rapid period of differentiation until E19, by which time a relatively stable adult-like pattern has been attained. The present findings indicate that many of the cyto- and chemoarchitectural features of this nucleus are present well before birth, by which time the nucleus must serve vitally important functions such as relaying information for control of respiration and the circulation. Accepted: 24 October 2000     

Calcium-contraction relationship in rat mesenteric arterial smooth muscle. Effects of exogenous and neurogenic noradrenaline

 We evaluated the relationship between intracellular calcium concentration ([Ca²⁺]_i) and vasoconstriction during the presence of exogenous noradrenaline (NA) and sympathetic nerve stimulation. An imaging technique was used to determine calcium/tension relationships in isolated rat mesenteric resistance arteries that had been mounted for recording of isometric tension development and loaded with Fura-2/AM. Experiments were performed after depletion of vasodilator neuropeptides and in the continuous presence of 1 μM propranolol, 3 μM indomethacin, and 30 μM nitro-l-arginine. NA (10 μM) was shown first to induce a further increase in tension, but not [Ca²⁺]_i, during the contraction induced by 125 mM K⁺. Subsequently, calcium/tension relationships were determined during stimulation with graded increases in extracellular [K⁺] (5.9–125 mM K⁺), cumulative administration of NA (0.2–10 μM) and electrical field stimulation of perivascular nerves (EFS, 1–16 Hz). A basal calcium/tension relationship without the calcium-sensitizing property of NA was constructed using a cumulative concentration/response curve of 5.9–125 mM K⁺ in arteries after prior exposure to the irreversible α-adrenoceptor antagonist phenoxybenzamine (POB). K⁺ series before and during α-blockade were also studied using the combination of the α₁-antagonist prazosin and α₂-antagonist yohimbine yielding comparable results as with POB. Calcium/tension curves obtained in the presence of NA, K⁺ and during EFS all were shifted to the left compared with the basal condition and all showed a similar slope indicating that neurogenically released NA is equally capable of inducing calcium sensitization in smooth muscle of mesenteric resistance arteries as exogenously applied NA. In the presence of exogenous and endogenous NA we not only observed an elevated contractile response for a given increase in [Ca²⁺]_i, but also an attenuated rise in [Ca²⁺]_i for a given intensity of stimulation. This suggests that the agonist-induced calcium-sensitization is accompanied by a reduction of the rise in [Ca²⁺]_i. Received: 24 October 1997 / Received after revision: 26 January 1998 / Accepted: 26 February 1998     

Attenuation of myosin light chain phosphorylation and posttetanic potentiation in atrophied skeletal muscle

 Previously we have demonstrated that the absence of staircase potentiation in atrophied rat gastrocnemius muscle is accompanied by a virtual absence of phosphorylation of the regulatory light chains (R-LC) of myosin. It was our purpose in the present study to determine if posttetanic potentiation and corresponding R-LC phosphorylation were also attenuated in disuse-atrophied muscles. Two weeks after a spinal hemisection (T12), twitch and tetanic contractile characteristics were measured in situ in control, sham-treated and atrophied (hemisected) muscles. Posttetanic potentiation 20 s after a 2 s tetanic contraction (200 Hz) was depressed in atrophied muscles (128.7 ± 2.6%; mean ± SEM) when compared to sham-treated (149.9 ± 2.4%) and control (142.9 ± 2.7%) muscles. Atrophied muscles demonstrated a significant increase in R-LC phosphorylation from rest (0.05 ± 0.04 moles of phosphate/mole of R-LC) to posttetanic conditions (0.21 ± 0.03 moles of phosphate/mole of R-LC), and less phosphorylation than control and sham-treated muscles (0.43 ± 0.06 and 0.49 ± 0.03 moles of phosphate/mole of R-LC, respectively) after tetanic stimulation. The preservation of the potentiation-phosphorylation relationship in atrophied muscles is consistent with the hypothesis that R-LC phosphorylation may be the principal mechanism for twitch potentiation. Received: 19 June 1997 / Accepted: 17 July 1997     

Intraventricular injection of NGF, but not BDNF, induces rapid motor activation that is inhibited by nicotinic receptor antagonists

 The acute and subacute effects of intracerebroventricularly (ICV) administered nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) on locomotor activity were evaluated in awake adult rats. Immediately after ICV injection through an implanted cannula, locomotor activity was measured by a computerized system using infrared photocells, which allowed us to record locomotion, motility, and rearing simultaneously. A single dose of 5 μg mouse β-NGF produced significant increases in horizontal ambulatory components of locomotor activity (locomotion and motility), but not vertical movement (rearing) 30–45 min after ICV administration. These increases lasted for at least 3–4 h. Systemic injection of 2.0 mg/kg mecamylamine, a central nicotinic receptor antagonist, inhibited the hyperactivity induced by NGF. Systemic injection of 0.5 mg/kg scopolamine, a muscarinic receptor antagonist, did not interfere with the NGF effects. Thus, while scopolamine induced marked increases in all three measures of behavior in both NGF and cytochrome-c-treated animals, locomotion and motility remained significantly higher in the NGF group. Immunohistochemistry demonstrated that NGF diffused readily from the ventricular space into brain parenchyma on the injected side and could be visualized 1 h after ICV injection. These results suggest that ICV administration of NGF increases locomotor activity by inducing acetylcholine release, and that nicotinic receptors are involved in the hyperactivity induced by NGF. ICV administration of 5 μg recombinant human BDNF had no significant effect on locomotor activity during the 0- to 4-h period after ICV injection. However, it produced significant decreases in locomotion, motility, and rearing 24–26 h later. Hence ICV administration of BDNF has entirely different effects on animal behavior from those evoked by NGF. While NGF elicits increases in ambulatory behavior within hours, BDNF causes delayed decreases in ambulatory behavior. Received: 21 October 1996 / Accepted: 20 March 1997     

Comparative study of the neuronal plasticity along the neuraxis of the vibrissal sensory system of adult rat following unilateral infraorbital nerve damage and subsequent regeneration

 The aim of the present study was to examine the physiological consequences of a unilateral infraorbital nerve lesion and its regeneration at different levels of the somatosensory neuraxis. In animals whose right infraorbital nerve had been crushed, a large unresponsive area was found in the main brainstem trigeminal nucleus (Pr5). Responses evoked by ipsilateral vibrissal deflection in the middle of Pr5 reappeared only on days 22–35 after the nerve had been transected, whereas recovery from the nerve crush took only 7–9 days. However, no sign of short-term neuronal plasticity was observed in Pr5 after peripheral nerve injury. An enlargement of the receptive fields in two-thirds of the units and a lengthening in the delay of the evoked responses were observed as long-term plastic changes in Pr5 neurons after peripheral-nerve regeneration. In the ventral posteromedial nucleus of the thalamus (VPM) of partly denervated animals, however, only minutes or hours after the nerve crush, certain units were found to respond in some cases not only to the vibrissae, but also to mechanical stimulation of the face over the eye (two units), the nose (one unit), and the midline (one unit). Apart from the experiments involving incomplete denervation, the vibrissal representation areas of the VPM were unresponsive to stimulation of both the vibrissae and other parts of the face until nerve regeneration had occurred. In the somatosensory cortex, an infraorbital nerve crush immediately resulted in a large cortical area being unresponsive to vibrissal deflection. It was noteworthy, however, that shortly after the nerve crush, this large unresponsive whisker representation cortical area was invaded from the rostromedial direction by responses evoked by stimulation of the forepaw digits. In spite of the reappearance of vibrissa-evoked responses 7–10 days after the nerve crush, an expanded digital representation could still be observed 3 weeks after the nerve crush, resulting in an overlapping area of digital and vibrissal representations. The withdrawal of the expanded representation of forepaw digits was completed by 60 days after the nerve crush. The results obtained in Pr5, the VPM, and the cortex strongly suggest that the higher the station in the neuraxis, the greater the degree of plasticity after infraorbital nerve injury. Received: 28 May 1998 / Accepted: 5 January 1999     

Pulmonary neuroepithelial bodies are innervated by vagal afferent nerves: an investigation with in vivo anterograde DiI tracing and confocal microscopy

 The pulmonary airway and alveolar epithelia contain distinctly innervated clusters of basally granulated cells: the neuroepithelial bodies. In the past, morphological criteria and the results of selective vagotomy have led to the interpretation that their innervation is sensory. Consequently, they are regarded as receptor organs. As a further test of this hypothesis, the present investigation set out to label vagal sensory nerve fibres to the lungs by anterograde neural tracing, and to establish the relationship between these fibres and the neuroepithelial bodies. A fluorescent neural tracer was injected unilaterally into the left or right nodose ganglion of adult rats. After suitable survival times, thick frozen sections of lung tissue were studied with laser scan confocal microscopy. Sensory nerve fibres were seen to run in the airway walls and occasionally penetrated the epithelium, where they formed complex terminals. The resulting intraepithelial sensory end organs showed a close morphological resemblance to the neuroepithelial bodies. Subsequently, electron microscopic investigation of such identified structures revealed the typical ultrastructural characteristics of neuroepithelial bodies: corpuscular cells containing dense cored secretory vesicles and contacted by mitochondria-rich nerve endings. We conclude that anterograde tracing of sensory nerves from the nodose ganglion confirms the receptor nature of the pulmonary neuroepithelial bodies, which may correspond to a subpopulation of the irritant and C-fibre receptors. Accepted: 14 October 1997     

Infraterminal spreading and extrajunctional expression of nicotinic acetylcholine receptors in denervated rat skeletal muscle

 Through the use of biotinylated-bungarotoxin and monoclonal antibodies, the nicotinic acetylcholine receptor (nAChR) was localized in the subneural apparatus of mammalian motor end plates of the flexor digitorum brevis muscle of the adult rat at the light and electron microscopic levels. Under normal conditions, nAChR was located in the primary post-synaptic membrane of the neuromuscular junction, and the depths of the junctional folds constituting the secondary post-synaptic membrane did not contain any nAChR. Up to 75 days after repeated transection of the related motor nerve (sciatic), there was no major alteration in the light-microscopic localization of junctional nAChR in the subneural apparatus, except for a moderate shrinkage and increased immunocytochemical reactivity of the subneural apparatus. At the electron microscopic level, however, immunocytochemical reactivity gradually occupied the entire extent of the secondary post-synaptic membrane, including the depths of the junctional folds, which exhibited extensive branching. In non-innervated portions of the muscle fibers, nAChR receptor appeared in a linear localization on the surfaces of denervated muscle fibers. This linear reaction was not continuous with the nAChR reaction of the motor end plates. It is concluded that denervation supersensitivity might not be due to spreading of junctional nAChR from the end-plate area, but rather to expression of nAChR in non-innervated portions of the muscle fiber and to the infraterminal (subsynaptic) spreading of nAChR into the depths of junctional folds. Received: 29 May 1998 / Accepted: 8 December 1998     

Experimental model of trigeminal pain in the rat by constriction of one infraorbital nerve: changes in neuronal activities in the somatosensory cortices corresponding to the infraorbital nerve

 Neuronal activities of the somatosensory (Sm1) vibrissa cortex were explored bilaterally under moderate gaseous anaesthesia in rats with a chronic constriction injury (CCI) of an infraorbital nerve (IoN). The CCI-IoN rats exhibited abnormal pain-related reactions to mechanical stimuli directed to the territory of the injured nerve, just prior to the recording session. The responsiveness, laminar localisation, and somatotopic organisation of 388 neurones were recorded in the pad vibrissa cortex contralateral (Cc, n=249) and ipsilateral (Ci, n=139) to the injured nerve, analysed, and compared with 223 neurones recorded in a parallel study of the Sm1 cortex in normal rats (Cn). The mean background activity of all the recorded neurones was relatively low, whether they were located in the Cn, Cc or Ci. The responsive neurones occurred in similar proportions in the Cc and Ci (∼55%) and significantly more frequently than in the Cn (35%). They consisted mainly of vibrissa neurones (100% in Cn, 85% in Cc, and 93% in Ci). The ratio [vibrissa neurones/(vibrissa+unresponsive) neurones] was enhanced in all the cortical layers of the Cc and Ci, except in the Cc layer IV. The receptive field (RF) size of the vibrissa neurones also differed greatly. It was limited to one vibrissa for 88% of the Cn neurones, but expanded to two or more vibrissae for 72% of the Cc and 52% of the Ci neurones. These multivibrissa units, mostly located in layers V and VIa for the few Cn neurones, were scattered in the different layers in CCI-IoN rats, with the largest RFs occurring in the deepest layers. In parallel, the cortical somatotopy of the vibrissae, roughly comparable with that initially described in pioneer studies of normal rats, was dramatically disturbed not only in the Cc, but also in the Ci of CCI-IoN rats. Contrasting with the results previously obtained in the ventro-postero-median thalamic nucleus of CCI-IoN rats, no neurones were driven by pinches or pinpricks applied to the cutaneous part of the vibrissa pad. It is questioned whether the disorganisation within the cortical map of the whisker pad, and the expanded RFs of vibrissa neurones could account for the abnormal pain-related reactions elicited from the massively deafferented trigeminal area. Received: 21 September 1998 / Accepted: 19 January 1999     

Ischaemia-induced temporal expression of connexin43 in rat heart

 To investigate the regulation of cell-to-cell coupling in myocardial ischaemia, the three-dimensional expression of connexin43 (Cx43) during experimental ischaemia was examined using a confocal laser scanning microscope. After induction of myocardial infarction in rats, serial optical sections were obtained from the left ventricular myocardium at various times (3 h to 60 days after ligation). The expression of Cx43 was detected immunohistochemically with FITC-labelled anti-rat Cx43 antibody. Fluorescent dots of Cx43 remained along the intercalated disc and decreased in number around the infarct up to 12 h after ligation. Cx43-expression disappeared completely within 48 h after ligation. After day 4, and especially on days 8 and 15 after ligation, the edges of the cardiomyocytes bordering the infarcted area manifested numerous sarcoplasmic tentacles that reacted positively to anti-desmin antibody. Distinct expression of Cx43 was observed extensively on the tentacles, although no cardiomyocytes remained viable around them. By day 60 after ligation, atypical expression of Cx43 had disappeared. These findings suggest that ischaemia induces temporally abnormal expression of Cx43, which might be responsible for abnormal conduction around the infarct. Received: 4 April 1997 / Accepted: 19 June 1997     

Aversive effects of the C-fragment of Substance P in the dorsal periaqueductal gray matter

There has been an increasing interest in the role of neuropeptides in the integration of brain functions. Besides the well-known positive-reinforcing effects of Substance P (SP) in prosencephalic regions, a role of this neuropeptide in the generation of aversive states in mesencephalic structures has also been envisaged. Evidence from a previous study suggests an involvement of SP in the neural substrates of aversion in the dorsal periaqueductal gray matter (DPAG). In the present study, we investigate whether N- and C-terminal fragments of Substance P are responsible for the effects produced by microinjections of SP into the dorsal periaqueductal gray. The results show that SP and its C-terminal fragment SP_7–11 produced a behavioral activation with increases in locomotor activity, grooming, and rearings, while the N-terminal fragment SP_1–7 produced only an increase in vertical exploratory activity. The effects were more pronounced with intermediate doses of SP and its C-fragment, confirming the characteristic bell-shaped dose-effect function of this neuropeptide. The proaversive effects observed with DPAG microinjections of these neuropeptides in the present study gain further relevance when combined with previous reports showing unconditioned and conditioned aversive effects following DPAG microinjections of SP in the place aversion and the elevated plus maze tests, two widely used animal models of anxiety. These results confirm previous data showing that SP has a modulatory role in the DPAG and that its effects are probably due to its C-terminal fragment.