Immunolocalization of nitric oxide synthases at the postsynaptic domain of human and rat neuromuscular junctions--light and electron microscopic studies.

Neuronal (n) and inducible (i) nitric oxide synthase (NOS) were immunolocalized at the postsynaptic domain of human and rat neuromuscular junctions (NMJs) by light and electron microscopy. We applied polyclonal and monoclonal antibodies for colocalization with three other synaptic proteins, utilizing double and triple fluorescence labeling, and gold and peroxidase for immunoelectron microscopy. By light microscopy, nNOS and iNOS colocalized with desmin and dystrophin, known postsynaptic components, but not with neurofilament protein, a presynaptic component. By electronmicroscopy, nNOS, but not iNOS, colocalized postsynaptically on the same structures as desmin; iNOS was also postsynaptic, but did not colocalize with desmin immunoreactivity. At the NMJs of Duchenne muscular dystrophy patients, both nNOS and iNOS were strongly immunoreactive. At the NMJs of a patient with myasthenia gravis, nNOS was weaker than in controls. Total denervation of rat sciatic nerve did not cause any decrease of nNOS or iNOS immunoreactivity 7 days thereafter. At 15 days after denervation, there was a gradual decrease of immunoreactivity, and immunoreactivity disappeared 30 days after denervation, corresponding to the ultrastructurally detectable disorganization of the postsynaptic region. This seems to be the first combined light and electron microscopic description of the postsynaptic localization of nNOS and iNOS at human and rat NMJs.

Localization of Activin and Activin Receptors in Rat Spinal Motoneurons.

The purpose of this experiment was to determine whether rat spinal motoneurons (a) produce activin protein and (b) transcribe mRNAs coding for the betaA-subunit of activin and activin receptors II and IIB. The production of activin was determined by immunocytochemistry. The expression and localization of the mRNAs were elucidated by the reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization techniques. We have observed that activin A protein was produced and mRNAs encoding activin betaA-subunit and activin receptors II and IIB were expressed by motoneurons of the rat spinal cord. Furthermore, the identity of RT-PCR products was confirmed by DNA sequencing. It is concluded that activin may have a functional role in the maintenance of rat spinal motoneurons. Copyright 1996 S. Karger AG, Basel

Down-regulation of thyrotropin-releasing hormone (TRH) receptors in spinal cord after transection as revealed by quantitative autoradiography.

The autoradiographic localization of thyrotropin-releasing hormone (TRH) receptors was investigated in the rat spinal cord after transection at the level of T8-T9. The discrete distribution of [3H]-MeTRH binding was measured with a computerized image analyzer at the cervical (C6-C7) and lumbar (L2-L3) level, one week and three weeks after injury. The TRH receptor density was expressed in fmol/mg protein. There was no significant change in the density of TRH receptors below the injury site. In the cervical region, TRH receptor concentration in the dorsal gray matter did not differ from normal controls; in contrast we found a time dependent change in lamina 10 and in the ventral gray, with a significant decrease (25% and 19%, respectively) of TRH receptor binding sites one week after transection and a return to control levels by three weeks. From these data and the known increase of TRH immunoreactivity above a spinal injury, a down-regulation of spinal cord TRH receptors in response to elevated levels of TRH is suggested.

Effects of experimental spinal cord transection on substance P receptors: a quantitative autoradiography study.

The autoradiographic localisation of Substance P (SP) receptors was investigated in the rat spinal cord following cordotomy at the thoracic 8-9 level. The binding of [125I]-BH-SP was measured in discrete gray matter structures above (C6-C7 level) and below (L2-L3 level) the injury site, and expressed in fmol/mg protein. There was a statistically significant increase in SP receptor density 1 week after cordotomy in the dorsal horn and in the central canal of lumbar region, in laminae 3-4-5 and also in the ventral horn of cervical spinal cord. Elevated SP receptor density was also noted in cervical ventral horn at 3 weeks after thoracic cordotomy, whereas the increase seen at 1 week was normalized at 3 weeks everywhere else. Since SP concentration has been reported to show a similar localized increase below a spinal transection, the present results are consistent with an up-regulation of SP receptors. A similar direct relationship between SP receptors and SP content appears to occur also in the ventral horn above the injury site.

Response to deep hypoglycemia does not involve glucoreceptors in carotid perfused tissue.

In the present study we examined whether the magnified hormonal counter-regulatory response seen during deep hypoglycemia (40 mg/dl) could be attenuated by supplying the forebrain with glucose furnished through carotid infusion. Two protocols were performed in conscious dogs. In the first protocol we infused glucose bilaterally into the carotid circulation to produce a forebrain glycemia of 55 +/- 1 mg/dl (as reflected in the jugular vein), whereas systemic glycemia declined to 39 +/- 2 mg/dl. In the second protocol as a control we infused glucose into the systemic circulation at a rate matched to protocol 1 so that both systemic and jugular plasma glucose concentrations were equivalent to the systemic glucose concentrations in protocol 1 (jugular, 41 +/- 3 mg/dl; systemic, 40 +/- 2 mg/dl; P greater than 0.9). In spite of a substantial difference in forebrain glycemia (55 mg/dl compared with 41 mg/dl) there were no differences in the counter-regulatory responses of catecholamines or glucagon. In addition, through the use of radiolabeled microspheres, we defined the precise regions of the forebrain irrigated during bilateral intracarotid glucose infusions. The concentration of microspheres was high in the forebrain but very low in the hindbrain. Our results indicate that glucoreceptor cells in tissues perfused by carotid arteries may play a tautological role in the sympathetic response to hypoglycemia and imply that glucose-sensitive receptors must also be located elsewhere in the central nervous system or in the periphery.

Sinoaortic denervation changes catecholamines in caudate-putamen of rats.

The 3-day sinoaortic-denervated (SAD) rat has a reduced concentration of dopamine (DA) in caudate-putamen (CP) (Brain Research, 299 (1984) 380-383). This suggested that the nigrostriatal system processes input from the cardiovascular system via arterial baroreceptor pathways. In this study we compared regional DA levels in CP of SAD and sham-operated (SO) rats 3 and 7 days after surgery. Bilateral CP samples were obtained by micropunch from consecutive frozen brain sections. Samples were taken throughout the entire length of CP from dorsal, lateral and ventral areas. Punches of two consecutive sections were pooled and assayed for DA and norepinephrine (NE). SO rats showed a rostro-caudal gradient of DA in all 3 areas. Three-day SAD rats had significantly lower cumulative DA concentrations in each of the 3 CP areas. The concentrations in each region of the 3 areas were also reduced and about half of them achieved statistical significance. SO rats did not show a gradient of NE. Cumulative NE was significantly lower in dorsal and ventral areas of SAD rats. In 7 days SAD rats, DA values were no longer reduced, but were significantly elevated in dorsal but unchanged in ventral areas. These data support evidence of nigrostriatal interaction with baroreceptor pathways.

Autoradiographic localization of substance P receptors in rat spinal cord: effects of experimental spinal transection.

The autoradiographic localization of Substance P (SP) receptors was studied in the rat spinal cord following thoracic cordotomy. The binding of [125I]-BH-SP was measured in discrete gray matter structures above and below the injury site. There was a significant increase in SP receptor density 1 week after cordotomy in the dorsal horn and in the central canal of lumbar region, and in laminae 3-4-5 and also in the ventral horn of cervical spinal cord. Elevated SP receptor density was noted only in cervical ventral horn at 3 weeks after cordotomy. Since SP concentration has been reported to show similar localized increases, the present results are consistent with an up-regulation of SP receptors.

Analogs of thyrotropin-releasing hormone: hypotheses relating receptor binding to net excitation of spinal lower motor neurons.

Experimentally and clinically, treatment with high-doses of TRH produces a net excitation of spinal lower motor neurons (LMNs) that is subsequently reduced or completely lost through continuous or repeated exposure to the peptide. This is operationally termed "autorefractoriness" (AR). We have performed biochemical and in vivo pharmacologic experiments to investigate the mechanism(s) of AR. Biochemically, we classified TRH and several analogs into three groups based on their binding by spinal-cord TRH-receptors (TRH-Rs): high-affinity, (low nanomolar range; MeTRH, TRH); intermediate-affinity (mid-nanomolar range; MK-771, RX77368) or low-affinity (micromolar range; DN-1417, PNP). When tested in vivo for LMN excitatory activity in cordotomized (T8) rats, TRH and MK-771 produced rapid-onset excitation followed AR. In contrast, sustained excitation with much less AR was produced by the low affinity analog DN-1417. Based on these results, we have formulated two receptor-based hypotheses to explain AR: a) rapid TRH-R desensitization (conversion to an inactive form) by high- but not low-affinity TRH-analogs; and b) a slower down-regulation (cellular internalization) of the agonist-receptor complex, most evident with high-affinity agonists. Thus, low-rather than high-affinity TRH-analogs may be superior to TRH for providing sustained LMN excitation (increase of strength) in motor neuron degenerative disorders.

Pulmonary edema and death induced by sinoaortic denervation in fastigial nucleus-lesioned rats.

When adult male rats with lesions of the cerebellar fastigial nucleus were subjected to sinoaortic denervation and instrumented for aortic pressure recording, their elevated mean arterial pressure was found to rise no higher than that of rats with sinoaortic denervation alone; however all of the doubly operated rats died or became moribund within 4 days. Pulmonary edema and gastric ulcers were frequently seen. When the order of operations was reversed, all animals survived. The possible mechanism and involvement of other brain nuclei, catecholamines and vasopressin in these pathological changes is discussed.

Cardiovascular responses to central catecholamines of sinoaortic denervated rats.

The characteristic movement-related arterial pressure dips seen in sinoaortic-denervated (SAD) animals suggested they should have enhanced vasodepressor responses to central adrenergic agonists. This was tested by comparing the reductions in mean arterial pressure and heart rate of conscious sham-operated (SO) and SAD rats during injections of norepinephrine (NE) or epinephrine (E) (0.25--16 micrograms) into the fourth cerebral ventricle. NE produced significantly greater maximum vasodepression and, simultaneously, significantly less bradycardia in SAD than SO rats. E, like NE, produced significantly more vasodepression in SADs, but unlike NE, elicited the same magnitude of bradycardia in both groups. The pressures were reduced and the log-dose pressure response curves shifted to the left in SAD rats. These results provide indirect evidence that central sensitivity to intracerebroventricular catecholamines is altered in SAD rats and that the direction of change in sensitivity is divergent for pressure and heart rate.

Effects of an intraventricular injection of synthetic ACTH on plasma testosterone, progesterone and LH levels and on sexual behavior in male and female rabbits.

Sexual behavior and elevation of gonadal steroids were induced in both male and female New Zealand white rabbits following injection of synthetic beta (1-24) ACTH into the lateral cerebral ventricle. In blood assays collected by cardiac puncture, testosterone, progesterone and corticosterone were all increased above both resting levels and samples were taken after a control intraventricular injection of physiological saline. Parallel increases of plasma LH were also demonstrated. The results were interpreted to indicate that intraventricular ACTH, through LH release, influences sex hormone levels and sexual behavior by 'centrally', probably within the hypothalamus.