Hyperpolarization of hypothalamic parvocellular neurons by 17 β-estradiol and their identification through intracellular staining with procion yellow

Summary Intracellular recordings and injections of procion yellow (PY) were made in parvocellular neurons in hypothalamic slices of female guinea pigs. Eighty-five neurons, with an average resting membrane potential of -35 mV, were recorded in the arcuate (ARC) ventromedial (VM), and in the cellpoor zones between the ARC and VM. Eleven of the ARC neurons and four neurons from the cell-poor zone could be driven antidromically by median eminence (ME) stimulation, nine other neurons from the three areas could be driven orthodromically by stria terminalis (ST) stimulation.Twenty-eight parvocellular neurons were tested with 17 -estradiol (E₂), which was applied in the bathing medium as the free steroid. Eleven neurons (nine ARC and two cell-poor-zone neurons) were hyperpolarized 2 to 24 mV by 10^–10 M E₂ concentrations. 10^–8 M estrone concentration was without effect on three of these cells. Through the intracellular injection of PY, the estrogen-sensitive neurons (N = 11) were identified as small fusiform cells with few dendrites. Spine-like appendages were found on only one of these cells. None of the larger pyramidal-like neurons of these areas responded to the application of E₂.Postdoctoral research fellow of the National Institute of Neurological and Communicative Disorders and Stroke     

“Dark” (compacted) neurons may not die through the necrotic pathway

Dark neurons were produced in the cortex of the rat brain by hypoglycemic convulsions. In the somatodendritic domain of each affected neuron, the ultrastructural elements, except for disturbed mitochondria, were remarkably preserved during the acute stage, but the distances between them were reduced dramatically (ultrastructural compaction). Following a 1-min convulsion period, only a few neurons were involved and their environment appeared undamaged. In contrast, 1-h convulsions affected many neurons and caused swelling of astrocytic processes and neuronal dendrites (excitotoxic neuropil). A proportion of dark neurons recovered the normal structure in 2 days. The non-recovering dark neurons were removed from the brain cortex through two entirely different pathways. In the case of 1-h convulsions, their organelles swelled, then disintegrated and finally dispersed into the neuropil through large gaps in the plasma membrane (necrotic-like removal). Following a 1-min convulsion period, the non-recovering dark neurons fell apart into membrane-bound fragments that retained the compacted interior even after being engulfed by astrocytes or microglial cells (apoptotic-like removal). Consequently, in contrast to what is generally accepted, the dark neurons produced by 1-min hypoglycemic convulsions do not die as a consequence of necrosis. As regards the case of 1-h convulsions, it is assumed that a necrotic-like removal process is imposed, by an excitotoxic environment, on dark neurons that previously died through a non-necrotic pathway. Apoptotic neurons were produced in the hippocampal dentate gyrus by intraventricularly administered colchicine. After the biochemical processes had been completed and the chromatin condensation in the nucleus had reached an advanced phase, the ultrastructural elements in the somatodendritic cytoplasm of the affected cells became compacted. If present in an apparently undamaged environment such apoptotic neurons were removed from the dentate gyrus through the apoptotic sequence of morphological changes, whereas those present in an impaired environment were removed through a necrotic-like sequence of morphological changes. This suggests that the removal pathway may depend on the environment and not on the death pathway, as also assumed in the case of the dark neurons produced by hypoglycemic convulsions.     

Participation of Ia reciprocal inhibitory neurons in the spinal circuitry of the tonic neck reflex

Summary As part of our studies of the spinal circuitry of the tonic neck reflex, we have recorded extracellularly from Ia reciprocal inhibitory neurons of the decerebrate, labyrinthectomized cat. The activity of a majority of neurons driven by stimulation of the quadriceps nerve was modulated by sinusoidal rotation of the neck; such modulation was much less frequent in the case of neurons driven by stimulation of nerves to more distal muscles. The results suggest that some of the inhibition which is part of the tonic neck reflex is mediated by Ia reciprocal inhibitory neurons, but that other pathways must also play an important role.     

Developing neuronal populations of the cat retinal ganglion cell layer

An improved flat-mount procedure demonstrates that the developing ganglion cell layer of the cat retina contains two morphologically distinct populations of presumed neurons at all ages between embryonic day 36 (E36) and adulthood. One population resembles the adult "classical neurons" composing the ganglion cells and bar-cells of Hughes, while the remaining cells, which are smaller and possess much less Nissl substance, presumably correspond to precursors of the adult microneurons. Although the total neuron population of the retinal ganglion cell layer remains quite constant at all studied ages, its component subpopulations alter significantly during prenatal development; some 50% of classical neurons disappear before birth and the microneuron population doubles during the same period. An obvious centroperipheral gradient exists for classical neurons by stage E47, but the microneuron density gradient only becomes apparent at birth. A 2:1 centroperipheral ratio for the total neuron population is also apparent at E47. Centroperipheral neuronal density gradients continue to increase during postnatal growth. Loss of classical neurons during prenatal life as a result of cell death or transformation into microneurons, has been postulated as a mechanism for determining neuron density gradients. Cell death does occur in the ganglion cell population but it is not yet established whether microneurons of the ganglion cell layer originate from ganglion cell transformation or migrate as a differentiated class from the ventricular layer. However, it can be concluded that not all microneurons originate from ganglion cell transformation, because the total loss of classical neurons is less than the increase in microneuron numbers during development. The population magnitudes of both neuronal classes in the ganglion cell layer stabilise after birth. However, it is during the postnatal period that the adult cruciate density topography is achieved by both populations. It is concluded that differential areal growth is the prime mechanism for postnatal cell redistribution.     

Histological characterization of neurones innervating functionally different muscles of Locusta

The innervation of four functionally different muscles (subalar, remotor 1, remotor 2, pleuroalar), all served by the same nerve branch, was studied in both winged segments of the locust, Locusta migratoria. Several anatomical techniques were applied: With the cobalt backfill and silver intensification technique four cell types (motoneurone, dorsal unpaired median neurone, common inhibitory neurone, and small median neurone) were demonstrated. Serial sections enabled the morphology of the motoneurones to be described in more detail and in respect to a possible functional organization of the arborizations. A differential staining technique allowed us to stain various neurones in different colours in the same preparation. With this technique the anatomy of both the "rostral" and the "caudal" subalar motoneurones could be described in parallel, thus avoiding errors in comparison due to possible individual variations from preparation to preparation. Axon counts in the peripheral nerve branch enabled us to compile a list of the total innervation for each muscle. Results from other orthopterans are integrated and whether differences in the dendritic fields might be of functional significance is discussed.     

Endogenous dopamine release from tuberoinfundibular neurons: Does calmodulin play any role?

Summary The possible involvement of calmodulin in the process of endogenous dopamine (DA) release from arcuateperiventricular nuclei-median eminence fragments, containing tuberoinfundibular dopaminergic (TIDA) neurons, has been investigated in an in vitro incubation system. For this purpose the basal and K⁺-stimulated DA release was examined in the presence and in the absence of the different putative calmodulin antagonists, pimozide, trifluoperazine, penfluridol and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7).Trifluoperazine and pimozide in concentrations up to 100 M were both uneffective in blocking K⁺-evoked DA release. Penfluridol in doses of 5 and 10 M, did not prevent 35 mM K⁺-induced endogenous DA release. It was able to reduce K⁺-stimulated DA release only at the very large concentration of 100 M.W-7 added in vitro to the hypothalamic fragments, prevented endogenous DA release evoked by 35 mM K⁺ in a dose-dependent manner. W-5, a chlorine deficient analogue of W-7, that interacts only weakly with calmodulin, failed to modify K⁺-stimulated endogenous DA release in doses up to 200 M.All the putative calmodulin antagonists used in the present study did not induce any change of basal DA release.IN conclusion the fact that most of the agents, except W-7, known to antagonize calmodulin-dependent processes in many biological systems failed to interfere with the release of endogenous DA from TIDA neurons seems to suggest that calmodulin does not play a crucial role in the process of DA release and that the inhibitory effect of W-7 on endogenous DA release may be better attributed to other mechanisms different from its anticalmodulin action.     

Effects of noradrenaline and somatostatin on basal and stimulated mucosal ion transport in the guinea-pig small intestine

Summary Noradrenaline (NA) and somatostatin (SOM) stimulate intestinal water and ion absorption and are found in mucosal nerve fibres and nerve terminals in submucous ganglia of the guinea-pig small intestine. As the main projection of submucous neurons is to the mucosa, NA and SOM might alter mucosal transport either by a direct effect on the epithelium or indirectly, by affecting submucous neurons. In this study these two possible sites of action of NA and SOM have been investigated in mucosa-submucosa preparations of guinea-pig ileum. In addition, the actions of NA and SOM on the secretory responses caused by stimulation of different populations of submucous neurons have been studied. The stimulants of secretion used were a nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP, 10^–5 M), 5-hydroxytryptamine (5-HT, 10^–7 M) and electrical field stimulation (EFS), which activate cholinergic, noncholinergic and mixed populations of submucous secretomotor neurons, respectively.Segments of intestine were dissected free of external muscle and myenteric plexus and mounted in Ussing chambers. Short-circuit current (I _sc) was measured as an indication of net active ion transport across the tissue. NA (10^–8 M) and SOM (>10^–10 M) each caused a decrease in I _sc, indicating a net increase in ion absorption. The NA response was abolished and the magnitude of the SOM response was reduced to 20% by tetrodotoxin (10^–7 M). DMPP, 5-HT and EFS each stimulated nerves that increased I _sc and each of these responses was significantly diminished by NA and SOM; for both NA and SOM the decrease in the DMPP response was significantly greater than the decrease observed in the response to carbachol (10^–6 M). Phentolamine (10^–6 M) abolished all of the effects of NA but caused no change in the SOM effects. These studies have shown that NA and SOM cause similar changes in net ion transport, that their actions are primarily on submucous secretomotor neurons and that NA and SOM can diminish the responses to stimulation of both cholinergic and noncholinergic submucous neurons.In this tissue it is also known that SOM coexists with NA in noradrenergic nerve terminals in the submucosa. However, when applied together, NA and SOM caused no greater decrement in the carbachol and 5-HT responses than would be predicted by adding the separate effects of NA and SOM. Hence there was no obvious interaction between NA and SOM effects on mucosal transport.     

Apamin distinguishes two types of relaxation mediated by enteric nerves in the guinea-pig gastrointestinal tract

Summary Eight smooth muscle preparations from the stomach, small intestine and large intestine of the guinea-pig were used to compare apamin's actions in reducing the effectiveness of transmission from enteric inhibitory nerves and in reducing responses to inhibitory agonists ,-methylene ATP, VIP and isoprenaline. The effects of apamin on inhibitory reflexes in the ileum and colon were also evaluated. Apamin had little or no effect on responses to VIP and isoprenaline in any region, but consistently and substantially reduced responses to ,-methylene ATP. Responses to stimulation of enteric inhibitory neurons were substantially reduced by apamin in the antrum circular muscle, ileum longitudinal and circular muscle, taenia coli and distal colon longitudinal muscle, but it was ineffective in the fundus circular muscle, proximal colon longitudinal muscle and distal colon circular muscle. It caused a small reduction of the relaxation of the ileal circular muscle caused reflexly by distension, but did not modify the similar descending inhibitory reflex in the circular muscle of the colon. It is concluded that apamin can be used to distinguish two types of non-noradrenergic transmission from enteric inhibitory nerves to gastrointestinal muscle. Furthermore, neither VIP nor ATP can be the sole transmitter chemical released from enteric inhibitory neurons throughout the gastrointestinal tract.     

Sizes, laminar and topographic origins of cortical projections to the major divisions of the red nucleus in the monkey

The retrograde transport of horseradish peroxidase was used to study the topographic and laminar origins of the cortical projections to the parvocellular and the magnocellular divisions of the red nucleus in Macaca mulatta and Macaca fascicularis. Approximately 90% of the corticorubral projection is directed to the parvocellular division of the nucleus. Corticoparvocellular (CRp) neurons are pyramidally shaped, are smaller in size than corticospinal neurons, and are more numerous. They are found principally in sublamina Va of cytoarchitectonic areas 4 and 6, and in moderate quantities in sublamina Vb of posterior area 8 and area 5. In areas 4 and 6, the cells are grouped in clusters of three to 15 neurons each and are arranged in cellular bands of varying rostrocaudal thickness which course mediolaterally. With respect to functionally defined zones, CRp neurons are found throughout the supplementary motor area and the precentral motor cortex. In addition, they are found in parts of areas 5, 6, and 24 that project to these cortical motor areas, and that are thought to have "premotor" or movement-programming functions. The corticomagnocellular (CRm) projection arises principally from cells in sublamina Vb of the precentral arm and leg areas (area 4), and from adjacent parts of posterior area 6, CRm cells are pyramidally shaped, and their size distribution is bimodal, with peaks that correspond, respectively, to the modal diameters of CRp and of corticospinal neurons. These results and those of previous studies suggest that CRm neurons are involved principally in the control of hand and foot movements, with little effect on more proximal musculature. The massive CRp projection, however, is clearly part of a large cerebrocerebellar communication system, with motor and/or movement programming functions that have yet to be clearly defined.     

Inhibitory dopamine receptors on sympathetic neurons innervating the cardiovascular system of the pithed rat

Summary Additional experimental evidence was obtained for an inhibitory function of prejunctional ₂-adrenoceptors and/or dopamine receptors located on noradrenergic neurons innervating the heart and resistance vessels of the pithed normotensive rat. Mixed ₂-adrenoceptor receptor agonists, differing in selectivity towards either receptor type, i.e. N,N-di-n-propyldopamine (DPDA), 2-N, N-di-n-propylamino-6, 7-dihydroxy-1,2,3,4-tetrahydronaphthalene (DP-6,7-ADTN), B-HT 920 and B-HT 933 (azepexole) were used.In pithed normotensive rats, DPDA (30 and 100 g/kg/min) dose-dependently inhibited the electrical stimulation-induced increase in diastolic pressure, but did not significantly affect the stimulation-evoked increase in heart rate. The inhibition exerted by DPDA was blocked by haloperidol and sulpiride (0.3 mg/kg of each), but not by yohimbine (1 mg/kg), indicating the involvement of dopamine receptors. In this respect, sulpiride and haloperidol were found approximately equipotent.DP-6,7-ADTN (10 and 30 g/kg/min) impaired both tachycardic and vasoconstrictor responses in a dose-dependent manner. Sulpiride (0.3 mg/kg) only partially restored the DP-6,7-ADTN-depressed stimulation-evoked increase in diastolic pressure, whereas yohimbine (1 mg/kg) alone was without effect. The combination of both antagonists completely prevented the inhibition caused by DP-6,7-ADTN. On the other hand, yohimbine (1 mg/kg), but not sulpiride (0.3 mg/kg), selectively antagonized the DP-6,7-ADTN-induced inhibition of stimulation-evoked tachycardia.B-HT 920 (1, 3 and 10 g/kg/min) very effectively reduced the increase in diastolic pressure and heart rate caused by electrical stimulation. Inhibitory dopamine as well as ₂-adrenoceptors participated in the vascular effects of B-HT 920, whereas ₂-adrenoceptors were only involved in the cardioinhibitory response to this agonist.B-HT 933 (0.6 and 1 mg/kg/min) dose-dependently reduced the stimulation-evoked increase in arterial pressure through selective stimulation of inhibitory ₂-adrenoceptors, dopamine receptors not taking a part.The results confirm and extend the observations that in addition to ₂-adrenoceptors inhibitory dopamine receptors are located on the sympathetic neurons connected with the arterial vasculature of the pithed normotensive rat. The sympathetic nerves innervating the rat heart do not contain inhibitory dopamine receptors; their activity only can be modulated by ₂-adrenoceptor stimulation. In the pithed normotensive rat, activation of prejunctionally located ₂-adrenoceptors more effectively inhibits the sympathetic activity directed to the heart than that to the resistance vessels.