An ultrastructural study of the synaptic contacts of α-motoneurone axon collaterals. I. Contacts in lamina IX and with identified α-motoneurone dendrites in lamina VII

Horseradish peroxidase (HRP) was injected intracellularly in triceps surae α-motoneurones. The axons and axon collaterals of these neurones were traced light and electron microscopically. Synaptic boutons of collaterals, in Rexed's lamina IX or in synaptic contact with HRP-stained motoneurone dendrites in lamina VII, were studied ultrastructurally. The boutons exhibited spherical synaptic vesicles and made synaptic contacts of two different types with HRP-stained α-motoneurone dendrites in lamina IX and VII, dendrites and cell bodies of large neurones in lamina IX, dendrites of unknown origin in lamina IX and with one cell body of a medium sized neurone in lamina IX. The observations are discussed in relation to earlier qualitative and quantitative studies on the synaptology of cat spinal α-motoneurones.     

An ultrastructural study of the synaptic contacts of alpha 1-motoneuron axon collaterals. II. Contacts in lamina VII

Horseradish peroxidase (HRP) was injected intracellularly in triceps surae alpha-motoneurons. The axons and axon collaterals of these neurons were traced light and electron microscopically. Synaptic boutons of collaterals in the ventral part of Rexed's lamina VII were studied ultrastructurally. The boutons exhibited spherical synaptic vesicles and made synaptic contacts with cell bodies and proximal dendrites of neurons assumed to be Renshaw cells and with dendrites of unknown origin. The observations are discussed in relation to earlier qualitative and quantitative studies on the other known synaptic contacts of the alpha-motor axons, both in the central and peripheral nervous system.     

Direct monosynatpic contacts between type-identified α-motoneurons in the cat

We have examined, at the light microscope level, putative direct synaptic interconnections, via motor axon collaterals, between type-identified triceps surae α-motoneurons labeled by intracellular injection of HRP. The results indicate that monosynaptic recurrent contacts can occur between synergist motoneurons (in this case, medial gastrocnemius to soleus, and vice versa), irrespective of motor unit type (type FF to type S, and vice versa).     

α2 and α1-adrenoceptors mediate opposing actions on parasympathetic neurons

We used intracellular recording methods to analyze the membrane responses to norepinephrine in cat vesical parasympathetic ganglia. In parasympathetic neurons, norepinephrine (NE) produces a membrane hyperpolarization, a membrane depolarization often accompanied by cell firing and a biphasic potential, a hyperpolarization followed by a depolarization. We found that the NE hyperpolarization is mediated through alpha 2-adrenoceptors while the NE depolarization is mediated through alpha 1-adrenoceptors. This situation is different than in sympathetic neurons where beta-adrenoceptors mediate a NE depolarization.     

Autoradiographic localization ofβ1 andα1-adrenoceptors in the midbrain and forebrain of normal and reeler mutant mice

The distribution of alpha-1 and beta-1 adrenoceptors has been studied in the midbrain and forebrain of normal and reeler mutant mice, using autoradiographic visualization of radioiodinated HEAT and ICYP, respectively. All cortical structures and nuclear groups of the murine forebrain and midbrain bind ICYP and HEAT. For each ligand, there is substantial regional variation in binding density and these variations tend to observe boundaries between nuclei or cortical regions or the stratification of cortical regions. Regional variations in binding densities are generally different for ICYP and HEAT. Binding sites for ICYP are distributed densely throughout all fields of the neocortex (particularl, layersI–III > VI) and paleocortex, the striatum, pallidum, substantia nigra and superficial strata of the superior colliculus. Dense concentrations of binding sites for HEAT in cortical structures, by contrast, are limited to frontal (all layers except IV) and anterior cingulate regions of the neocortex and, as with ICYP, the stratum lacunosum-moleculaire of the regio superior of the hippocampal formation. In subcortical structures, again in contrast to the pattern with ICYP, binding density is greatest in the principal nuclei of the dorsal thalamus and the septal nuclei. the regional binding patterns of both ICYP and HEAT in the reeler brain are identical to those in the normal animal. Differential laminar binding patterns within the neocortex are approximately inverted in the two genotypes, however. Thus, binding of ICYP is densest in an inner zone of the mutant, but in the outer 3 layers of the normal neocortex. Binding of this ligand is of relatively lower density in an outer zone of the mutant and in the inner 3 layers of the normal neocortex. Similar inversions are characteristics of the laminar binding patterns of HEAT in the frontal, primary sensory and associational cortical regions of the two genotypes where densest binding is encountered superficially in reeler but at deeper levels of the normal neocortex.     

Does α-motoneurone size correlate with motor unit type in cat triceps surae?

The cell bodies and first-order dendrites of α-motoneurones supplying different functional types of muscle units in the cat gastrocnemius (type FF, FR and S units) and soleus (type SOL-S units) muscles, were studied after intracellular injection of horseradish peroxidase.The SOL-S neurones had smaller values for cell body diameter in comparison with both the FF and FR neurones. The SOL-S neurones also had significantly thinner first-order dentrites than the FF, FR and S neurones. In the gastrocnemius pool the S neurones had smaller values for dendritic diameters than the FF and FR cells.The values for combined diameter of the first-order dendrites indicated that the dendritic trees of the FF and FR neurones are, on the average, larger than those of the S and SOL-S neurones. Furthermore, the relationship between the combined dendritic diameter and the mean soma diameter, indicated that a difference in relative scaling of soma and dendrites exists between the FF and FR neurones on the one hand and the S and SOL-S neurones on the other. Similar results were obtained also when relating the combined dendritic parameter εd^3/2 to the soma surface area. Although a certain statistical relation seems to exist between motoneurone size and motoneurone type, it should be emphasized, however, that the range of values for each parameter studied overlapped considerably between the different types of motoneurones.     

Microinjection of carbachol in the lateral hypothalamus produces opposing actions on nociception mediated by α1- and α2-adrenoceptors

Electrical stimulation of the lateral hypothalamus (LH) produces antinociception partially blocked by intrathecal α-adrenergic antagonists, but the mechanism underlying this effect is not clear. Evidence from immunological studies demonstrates that substance P-immunoreactive neurons in the LH project near the A7 catecholamine cell group, a group of noradrenergic neurons in the pons known to effect antinociception in the spinal cord dorsal horn. Such evidence suggests that LH neurons may activate A7 neurons to produce antinociception. To test this hypothesis, the cholinergic agonist carbachol was microinjected into the LH at doses of 63, 125 and 250 nmol and the resulting effects on tail-flick and nociceptive foot-withdrawal latencies were measured. All three doses significantly increased response latencies on both tests, with the 125-nmol dose providing the optimal effect. Intrathecal injection of the opioid antagonist naltrexone (97 nmol) partially reversed antinociception, but neither the α₂-adrenoceptor antagonist yohimbine nor the α₁-adrenoceptor antagonist WB4101 altered latencies. However, two sequential doses of yohimbine blocked LH-induced antinociception on both tests. In contrast, two sequential doses of WB4101 increased nociceptive responses on both the tail-flick and foot-withdrawal tests. These findings, and those of published reports, suggest that neurons in the LH activate spinally projecting methionine enkephalin neurons, as well as two populations of A7 noradrenergic neurons that exert a bidirectional effect on nociception. One of these populations increases nociception through the action of α₁-adrenoceptors and the other inhibits nociception through the action of α₂-adrenoceptors in the spinal cord dorsal horn.     

α2, α2A, α2B/2C-Adrenoceptor subtype antagonists prevent lipopolysaccharide-induced fever response in rabbits

Exogenous pyrogens, e.g., bacterial lipopolysaccharides (LPS), are thought to stimulate macrophages to release endogenous pyrogens, e.g., TNFα, IL-1 β, and IL-6, which act in the hypothalamus to produce fever. We studied the effect of different α₁⁻ and α₂-adrenoceptor subtype antagonists, applied intraperitoneally, on the febrile response induced by LPS in rabbits. Evidence was obtained that prazosin, an α₁⁻ and α_2B/2C-adrenoceptor antagonist; WB-4101, an α₁⁻ and α_2A-adrenoceptor antagonist; CH-38083, a highly selective α₂-adrenoceptor antagonist (α₂: α₁ > 2000); BRL-44408, an α_2A-adrenoceptor antagonist; and ARC-239, an α_2B/2C⁻ and also α₁-adrenoceptor antagonist, blocked the increase of colonic temperature of the rabbit produced by 2 μg/kg LPS administered intravenously without being able in themselves to affect colonic temperature. In addition, prazosin, WB-4101 and CH-38083 antagonized the fall in skin temperature that occurred at the time when the colonic temperature was rising in control animals injected with LPS. All these results suggest that norepinephrine, through stimulation of both α₁⁻andα₂⁻ (α_2A⁻andα_2B/2C⁻) adrenoceptor subtypes, is involved in producing fever in response to bacterial LPS.     

Influence of centrally administered α- and γ2-melanocyte-stimulating hormone on hypothalamic blood flow autoregulation in the rat

The effect of intracerebroventricularly (i.c.v.) administered α-melanocyte-stimulating hormone (MSH) and γ₂-MSH on hypothalamic blood flow autoregulation was studied in anesthetized rats at different levels of standardized arterial hypotension. Autoregulation was impaired upon i.c.v. administration of 5 γ g/kg γ₂-MSH while α-MSH caused no change.. Since this effect of γ₂-MSH wa identical to that produced by i.c.v. naloxone in the same model, γ₂-MSH may be a functional antagonist of central opioid mechanisms participating in the control of cerebral blood flow autoregulation.     

Characteristics of the β1- and β2-adrenergic-sensitive adenylate cyclases in glial cell primary cultures and their comparison with β2-adrenergic-sensitive adenylate cyclase of meningeal cells

The agonist specificity pattern of the β-adrenergic adenylate cyclase in glial primary cultures was not typical of either β₁- or β₂-adrenergic receptors. The dose-response curves for adrenaline did not correspond to simple mass action kinetics and their computer analysis suggests the presence of both β₁- and β₂-adrenergic-sensitive adenylate cyclase (58 ± 17% and 42 ± 17% respectively).Similar properties of β₁- and β₂-adrenergic-sensitive adenylate cyclases were found by computer analysis of the dose-response curves for isoprenaline in the presence of a constant concentration of practolol (a selective β₁ antagonist) ( 55 ± 10% and 45 ± 10% of β₁- and β₂-sensitive adenylate cyclase respectively).The curves for displacement of [³H]dihydroalprenolol by practolol confirm these results.For purpose of comparison, the β-adrenergic receptors of meningeal cells in cultures were subjected to similar analysis. The results clearly showed that these cells exclusively contained β₂-adrenergic receptors.     

Spatial working memory improvement by an α2-adrenoceptor agonist dexmedetomidine is not mediated through α2C-adrenoceptor

1. Aged α_2C-adrenoceptor knockout and wild type mice were used to investigate whether α_2C-adrenoceptors are involved in mediating the beneficial effects of α₂-adrenoceptor agonist, dexmedetomidine, on spatial working memory.

2. A win-stay task in the radial arm maze was used to dissociate the effects of dexmedetomidine on working vs. reference memory. In addition, the animals were tested in simple response habit learning in the T-maze.

3. Knockout mice made more working memory errors after the change of the baited arm in radial arm maze, but after training reached again as accurate level of performance as wild type controls. Dexmedetomidine 5 and 10 μg/kg alleviated the increase in spatial working memory errors after the change of the baited arm in knockout mice. Knockout and wild type mice performed equally well in T-maze, and dexmedetomidine had no effect on this simple response learning.

4. The present results indicate that α₂-adrenoceptor agonists have a selective effect on spatial working memory not only in monkeys but also in mice. Further, this study confirms our earlier finding that the presence of α_2C-adrenoceptors is not necessary for the spatial working memory enhancing effect of α₂-adrenoceptor agonists.

Progesterone, 5α-pregnane-3,20-dione and 3α-hydroxy-5α-pregnane-20-one in specific regions of the human female brain in different endocrine states

Post-mortem concentrations of progesterone, 5α-pregnane-3,20-dione (5α-DHP) and 3α-hydroxy-5α-pregnane-20-one (allopregnanolone) were measured in 17 brain areas and serum in five fertile and five postmenopausal women. Steroid concentrations were measured with radioimmunoassay after extraction of brain tissue with ethanol and purification with celite chromatography. There were regional differences in brain concentrations of all three steroids. The highest progesterone levels were noted in the amygdala, cerebellum and hypothalamus and the highest levels of 5α-DHP and allopregnanolone were seen in the substantia nigra and basal hypothalamus. Brain concentrations of all three steroids were significantly higher in the fertile women in luteal phase compared to their postmenopausal controls (P<0.01). In general, the study showed that there is a variation in brain concentrations depending on ovarian steroid production, indicating that the secretion pattern during the menstrual cycle is reflected in the brain. However, regional differences in brain steroid levels imply local mechanisms for steroid uptake and binding as well. Investigations of gonadal steroid distributions in the human brain might be of importance considering the actions of these steroids in the central nervous system. Such studies could provide information about physiological mechanisms, such as the ovulation, and also form a baseline for comparative studies of normal and pathological conditions involving steroids, for instance, catamenial epilepsy and the premenstrual tension syndrome.     

Sevoflurane neurotoxicity in neonatal rats is related to an increase in the GABAAR α1/GABAAR α2 ratio

Exposure of neonatal rat to sevoflurane leads to neurodegeneration and deficits of spatial learning and memory in adulthood. However, the underlying mechanisms remain unclear. The type A γ‐aminobutyric acid receptor (GABA_AR) is a target receptor for sevoflurane. The present study intends to investigate the changes in GABA_AR α1/α2 expression and its relationship with the neurotoxicity effect due to sevoflurane in neonatal rats. After a dose–response curve was constructed to determine minimum alveolar concentration (MAC) and safety was guaranteed in our 7‐day‐old neonatal rat pup mode, we conducted two studies among the following groups: (A) the control group; (B) the sham anesthesia group; and (C) the sevoflurane anesthesia group and all three groups were treated in the same way as the model. First, poly(ADP‐ribose) polymerase‐1 protein (PARP‐1) expression was determined in the different brain areas at 6 hr after anesthesia. Second, the expression of PARP‐1 and GABA_AR α1/GABA_AR α2 in the hippocampus area was tested by Western blotting at 6 hr, 24 hr, and 72 hr after anesthesia in all three groups. After 4 hr, with 0.8 MAC (2.1%) sevoflurane anesthesia, the PARP‐1 expression was significantly higher in the hippocampus than the other brain areas (p < .05). Compared with Groups A and B, the expression of PARP‐1 in the hippocampus of Group C significantly increased at 6 hr after sevoflurane exposure (216% ± 15%, p < .05), and the ratio of the α1/α2 subunit of GABA_AR surged at 6 hr (126% ± 6%), 24 hr (127% ± 8%), and 72 hr (183% ± 22%) after sevoflurane exposure in the hippocampus (p < .05). Our study showed that sevoflurane exposure of 0.8 MAC (2.1%)/4 hr was a suitable model for 7‐day‐old rats. And the exposure to sevoflurane could induce the apoptosis of neurons in the early stage, which may be related to the transmission from GABA_AR α2 to GABA_AR α1.     

A comparison of α2-adrenoceptor regulation in brain and platelets

Functional responses andα₂-adrenoceptor radioligand binding were studied in brain and platelets of rabbits under a variety of circumstances. The effects of oestrogen treatment and maturation were studied in female rabbits and of aging and amitriptyline treatment in male rabbits. No correlation was found between changes in brain and platelets either in response orα₂-adrenoceptor ligand binding under any of the conditions examined.     

Effects of diet and obesity on brain α1- and α2-noradrenergic receptors in the rat

The chronic feeding of a sweetened condensed milk/corn oil diet (CM diet) to adult male rats produced significant increases in body weight and levels of plasma insulin in 34% of the rats fed this diet with respect to chow-fed controls. Levels of alpha 1-noradrenergic receptor binding were lower (32%) in the hypothalamic ventromedial nucleus (VMN) of only those rats which became obese (DIO rats) with respect to both chow-fed controls and those rats which resisted the development of obesity on the CM diet (DR rats). Also, alpha 1-noradrenergic binding was inversely proportional to body weight gain in the VMN (r = -0.831). alpha 2-Noradrenergic receptors were 30-37% lower in both the DIO and DR rats in the dorsomedial nucleus and dorsal area of the hypothalamus, and the medial dorsal area and nucleus reuniens of the thalamus. The similar decreases in alpha 2-noradrenergic receptors in both the DIO and DR rats in these areas suggested that dietary factors alone were responsible for these changes. There were no significant differences from chow-fed rats for hypothalamic dopamine (D2) or beta-noradrenergic (beta 1- and beta 2-) receptors in either DR or DIO rats. These results indicate that VMN alpha 1-noradrenergic receptors co-vary with body weight and implicate a role for alpha 1-receptors in the VMN in the central neuronal regulation of body weight.     

Noradrenaline- and time-dependent changes in neocortical α2- and β1-adrenoceptor binding in dorsal noradrenergic bundle-lesioned rats

A 6-hydroxydopamine-induced lesion of the dorsal noradrenergic bundle (DNB) in rats markedly decreased neocortical noradrenaline concentration (NNC) by 72–100% as measured 1, 3 and 13 months after the lesioning procedure. The concomitant assessment of neocortical α₂- and β₁-adrenoceptor binding (NAAB and NBAB, respectively) usually indicated significant increases of 25–74% for these two variables. There were, however, cases of unchanged NAAB and NBAB which presumably reflected an incomplete DNB lesion and a consequent time-related, partial recovery of NNC. The results emphasize the potential for long-term sequelae of the DNB lesion, and the existence of a critical NNC threshold (10–30% of control NNC values) which modulates postsynaptic α₂ and β₁-adrenoceptor density.     

Synthesis, metabolism and levels of the neuroactive steroid, 3α-hydroxy-4-pregnen-20-one (3αHP), in rat pituitaries

The neuroactive steroid, 3α-hydroxy-4-pregnen-20-one (3αHP), is a metabolite of progesterone and a precursor of 3α-hydroxy-5α-pregnan-20-one (5αP3α; allopregnanolone). In addition to analgesic and anxiolytic effects by interaction with the GABA_A receptor complex, 3αHP regulates pituitary FSH secretion by rapid non-genomic interaction with the Ca²⁺-driven cell signaling mechanisms. Since gonadectomy and adrenalectomy do not result in elimination of 3αHP, and since there is the possibility of paracrine and/or autocrine regulation of FSH release, the capacity of pituitary cells to regulate levels (by synthesis, metabolism, and storage) of 3αHP was examined. Anterior pituitaries from random cycling female rats were incubated, either as fragments or as cultured cells, for 1, 4 or 8 h with ³H- or ¹⁴C-labeled progesterone. The steroid metabolites were identified by thin-layer chromatography, autoradiography, high pressure liquid chromatography (HPLC), derivatization and GC/MS. Pituitary cells actively converted progesterone to 3αHP along with 5αP3α, 5α-pregnane-3,20-dione, 20α-hydroxy-5α-pregnan-3-one, 3β-hydroxy-5α-pregnan-20-one, 5α-pregnane-3α(β), 20α-diols, 20α-hydroxy-4-pregnen-3-one, and 4-pregnene-3α(β), 20α-diols. The results indicate the presence of the following steroidogenic enzymes in anterior pituitary cells: 3α-hydroxysteroid oxidoreductase (3α-HSO), 20α-HSO, 3β-HSO, and 5α-reductase. The activities of 5α-reductase and 3α-HSO were approximately equal and greatly exceeded those of the other enzymes. After 8 h of incubation with 100 ng progesterone per pituitary, about 20% of the progesterone was metabolized and 3.18 ng of 3αHP had been formed. The accumulation of 3αHP increased approximately linearly with the time of incubation. Metabolism studies using [1,2,6,7-³H]3αHP showed that pituitary cells convert about 29% and 8% of the 3αHP to progesterone and 5αP3α, respectively, in 2 h. Specific radioimmunoassays determined 11.6 and 7.5 ng of 3αHP per pituitary, respectively, in 25- and 40-day-old non-cycling female rats; these concentrations of 3αHP were about 2–3-fold greater than those of progesterone in the same pituitaries. In older (80–100 days old) cycling rats, the levels of 3αHP were about 9.4 and 18.6 ng/pituitary at 13.00 h and 22.00 h, respectively, on the day of proestrus, while the concomitant circulating levels were 13.7 and 5.4 ng/ml. The results indicate a marked capacity of rat pituitary cells to synthesize the neuroactive and FSH regulating steroid, 3αHP, from progesterone, and in turn to metabolize 3αHP to the neurosteroid, allopregnanolone, and to progesterone. The studies suggest cyclic biosynthetic and metabolic pathways for 3αHP and other steroids in the pituitary. They also indicate that the regulation of FSH secretion by 3αHP may be (in part, or in whole) via paracrine or autocrine mechanisms.     

Effect of α2 adrenergic agents upon central etorphine antinociception in the cat

Systemic (s.c.) administration of α₂ agonists clonidine (25–100 μg/kg) or guanfacine (50–400 μg/kg) elicited antinociception as assessed by the cat tail-flick model and potentiated in a dose-dependent manner the antinociceptive effect of etorphine (2.5 μg) administered directly into the periaqueductal gray. Conversely, systemic yohimbine (1 mg/kg) attenuated the effects of central etorphine, and diminished potentiation of etorphine by theα₂ agonists. Prior microinjection of clonidine (5μg) or guanfacine (5 μg) into the locus coeruleus (LC) reduced the intensity of central etorphine antinociception whereas central yohimbine (20 μg) pretreatment increased peak antinociceptive activity and prolonged the duration of etorphine. Thus, systemicα₂ agonists are inherently antinociceptive and potentiate central narcotic antinociception; however, the site of interaction betweenα₂ agonists and opiates does not appear to be the LC inasmuch asα₂ agonists attenuate the antinociceptive effect of etorphine when administered directly into the LC. A spinal site of action is suggested based upon known LC-spinal projections and our experimental observations.     

A role for central postsynapticα2-adrenoceptors in glucoregulation

Central or peripheral administration of theα₂-adrenoceptor agonist clonidine causes marked hyperglycemia in the rat. It is not clear whether this effect is mediated within the brain at either pre- or postsynapticα₂-adrenoceptors or whether it is due to peripheralα₂-agonist actions. We employed computerized mass spectrometry to measure noradrenaline (NA) and its primary neuronal metabolite 3,4-dihydroxyphenylglycol (DHPG) in the medial basal hypothalamus of rats treated acutely with clonidine, theα₂-antagonist yohimbine, the postganglionic noradrenergic blocker guanethidine and the neuroglycopenic agent 2-deoxy-d-glucose (2-DG). That clonidine's hyperglycemic effect was due, in part, to an action at centralα₂-adrenoceptors was indicated by the ability of guanethidine to significantly inhibit the glucose response. Because of clonidine's inhibition of hypothalamic NA release (assessed by the DHPG/NA ratio), presumably by presynaptic agonism, these data indicated that postsynaptic receptor stimulation by clonidine was involved in activating glucose release. Yohimbine markedly increased the hypothalamic DHPG/NA ratio, reflecting presynaptic stimulation of NA release, but at the same time inhibited the hyperglycemic response due to 2-DG administration. This latter effect to block hyperglycemia is consistent with antagonism of postsynapticα₂-adrenoceptors involved in mediating hepatic glucose output. These data indicate a major role for postsynapticα₂-adrenoceptors in glucoregulation.     

α2-Adrenergic receptors inhibit catecholamine secretion from bovine adrenal medulla

Catecholamine secretion evoked by carbymlcholine from isolated bovine adrenalinal medullary cells was inhibited byα ₂-agonist, clonidine, in a dose-dependent manner with IC₅₀ value of 2.8 × 10⁻⁵ M. [³H]Clonidine bound to adrenal medullary membranes with high affinity and saturable characteristics. These results suggest thatα ₂-adrenergic receptors which exist on adrenal medullary cells have inhibitory effect on the secretion of catecholamine from the cells