William E. Armstrong

A versatile means of intracellular labeling: injection of biocytin and its detection with avidin conjugates

Biocytin is a biotin-lysine complex of low molecular weight containing about 65% biotin, which retains a high affinity for avidin. Since the latter molecule has been conjugated to several histochemical markers, the use of biocytin as an intracellular marker was investigated. Electrodes were filled with a solution of 4-6% biocytin dissolved in 0.5 M KCl and 0.05 M Tris buffer, pH 7-7.6. Neurons were recorded intracellularly in the supraoptic nucleus of an explant preparation of the rat supraoptico-neurohypophysial system and injected for 1-20 min with either hyperpolarizing or depolarizing current. Following variable recovery times, the explants were fixed in either 10% formalin or 4% paraformaldehyde overnight, sectioned on a vibratome, and incubated with the avidin-biotin complex (ABC) or avidin which had been conjugated to fluorescein, rhodamine, Texas Red or horseradish peroxidase and containing 1% Triton-X 100. A high percentage of injected neurons were recovered using each of the labels with about equal success. Both negative or positive current injection could be used with little electrode clogging. Labeling with fluorescent conjugates was qualitatively similar to that of Lucifer Yellow, whereas labeling with avidin coupled to horseradish peroxidase or with ABC was qualitatively similar to filling neurons directly with horseradish peroxidase. The advantages of this technique are the ease of injection of biocytin and the versatility in allowing the investigator to choose among light-emitting and light-absorbing images.

Subnuclei in the rat hypothalamic paraventricular nucleus: a cytoarchitectural, horseradish peroxidase and immunocytochemical analysis.

Abstract A cytoarchitectonic scheme for the rat paraventricular nucleus has been proposed utilizing the retrograde transport of horseradish peroxidase, immunocytochemical localization of neurophysin-positive cells and fibers, Golgi-like impregnation of neurons, and observations of cell clustering as viewed in Nissl-stained material. Beginning rostrally, it was observed that the anterior commissural nucleus, a cell group sometimes claimed to be part of the paraventricular nucleus, contained many magnocellular perikarya which: 1. (1)projected to the neurohypophysis but apparently not to the brainstem or spinal cord; 2. (2) contained neurophysin; 3. (3) had few (one or two) dendrites, most of which projected toward the third ventricle; and 4. (4) were actually separated from the paraventricular nucleus by 300–400 μm. Proceeding caudally, the medial and lateral magnocellular portions of the paraventricular nucleus exhibited mostly the same characteristics as listed for the anterior commissural nucleus, except that the cells were slightly larger and more closely packed; these latter two form the conventional paraventricular neurosecretory nucleus. Cells of the dorsomedial cap and extreme posterior paraventricular nucleus: 1. (1) did not appear to project to the neurohypophysis, but did connect with the brainstem and spinal cord; and 2. (2) often contained neurophysin. In addition, large cells of the extreme posterior subnucleus: 1. (1) were more fusiform than neurons in the other three regions; 2. (2) possessed more (two or three) and longer dendrites than cells of the other divisions; and 3. (3) were loosely packed. Although some zones of overlap were noted, the results suggest that neurophysin-containing cells of the paraventricular nucleus are relatively segregated into neurohypophysial-projecting and brainstem/ spinal cord-projecting groups. This topographic separation and the morphological differentiation of these putative subnuclei suggest that these subdivisions may be controlled independently and that release of hormone from the neurohypophysis does not necessarily imply a coincidental release of hormone into other extrahypothalamic sites.

A biotin-containing compound N-(2-aminoethyl)biotinamide for intracellular labeling and neuronal tracing studies: Comparison with biocytin

The hydrochloride salt of a new, small molecular weight (M.W. = 286) biotin-containing compound referred to as biotinamide (N-(2-aminoethyl)biotinamide) was compared with biocytin (M.W. = 372) for its use in intracellular labeling of neurons and in neuronal tracing experiments using avidin conjugates for histochemical detection. The DC resistance and current passing ability of electrodes filled with 1-2 M potassium chloride, potassium acetate or potassium methylsulfate and containing 1-4% of these compounds were compared. Although differences were observed due to the electrolyte, with KCl electrodes being the least resistant, no electrode differences could be attributed to the concentration or type of tracer. However, whereas biocytin could be electrophoresed with either positive or negative current with roughly similar facility, biotinamide was selectively ejected with positive current. This would be beneficial to electrophysiologists using hyperpolarizing current to stabilize the membrane potential of neurons prior to recording. In addition, biotinamide-HCl could be dissolved at concentrations of 2-4% in either 1 or 2 M salt without precipitation, whereas biocytin precipitated in some of these solutions. Both compounds were equally useful for neuronal tracing experiments with survival times of 2 days, but labeling was much weaker with longer survival times. There was also little difference in the ability to histochemically localize these compounds using avidin conjugates, including avidin-biotin-horseradish peroxidase complex. In conclusion, biotinamide shares many of the useful features of biocytin, but can be selectively electrophoresed with positive current and can be dissolved at higher concentrations with little detriment in the electrical properties of the recording electrode.

Morphological and electrophysiological classification of hypothalamic supraoptic neurons.

In mammals, the magnocellular neurons of the supraoptic nucleus (SON) have been classified into vasopressin- (VP) and oxytocin- (OT) producing subtypes. The degree to which these neurons have distinguishable characteristics is considered in the present review. Most of the cytoarchitectonic diversity observed in some Golgi studies has yet to be attributed to differences between OT and VP neurons. The predominant SON cell type is a large bipolar neuron with relatively short and simply branching dendrites. Based on intracellular filling, large multipolar neurons probably represent a small subset of neurosecretory cells. Parvicellular multipolar and bipolar neurons may represent interneurons or subsets of neurosecretory cells. Suggestive evidence that axonal origin and spine density may differ between OT and VP neurons remains to be confirmed in rat. Different fiber systems are thought to preferentially innervate VP or OT subgroups, but only rarely have inputs to OT and VP neurons been compared at the ultrastructural level. Potentially selective inputs to OT somata may derive from the raphe system and the nucleus of the solitary tract, whereas the apparent preferential innervation of VP neurons (e.g. from the A1 region of the ventrolateral medulla) is less certain because of the overlapping dendritic fields of OT and VP neurons. Electrophysiologically, OT and VP neurons are best distinguished in vivo by their reaction to gastric, cardiovascular and suckling stimuli. The firing patterns of activated OT and VP neurons often differ, but can transiently appear indistinguishable in vivo and especially in vitro. Classification in vitro without immunochemical labelling may be aided by the presence of phasic bursting (mostly in VP neurons) and by the differential response of these neurons to certain neurochemicals or to stimulation of certain inputs. The membrane properties of OT and VP neurons are generally similar in vitro, but the range of tests has not been extensive. The depolarizing afterpotential is more often exhibited by, but is not exclusive to, VP neurons.

Extra-hypothalamic afferent inputs to the supraoptic nucleus area of the rat as determined by retrograde and anterograde tracing techniques

To detect neuronal cell bodies whose axon projects to the hypothalamic supraoptic nucleus, small volumes (10-50 nl) of 30% horseradish peroxidase or 2% fast blue solutions were pressure-injected into the area of one supraoptic nucleus of rats. Both dorsal and ventral approaches to the nucleus were used. In animals where the injection site extended beyond the limits of the supraoptic nucleus, retrogradely labelled cell bodies were found in many areas of the brain, mainly in the septum, the nucleus of the diagonal band of Broca and ventral subiculum in the limbic system; the dorsal raphe nucleus, the locus coeruleus, the nucleus of the dorsal tegmentum, the dorsal parabrachial nucleus, the nucleus of the solitary tract and the catecholaminergic A1 region in the brain stem; in the subfornical organ and the organum vasculosum of the lamina terminalis, as well as in the median preoptic nucleus. In contrast, when the site of injection was apparently restricted to the supraoptic nucleus, labelling was only clearcut in the two circumventricular organs, the median preoptic nucleus, the nucleus of the solitary tract and the A1 region. Injections of wheat germ agglutinin coupled with horseradish peroxidase (60-80 nl of a 2.5% solution) made in the septum and in the ventral subiculum anterogradely labelled fibers coursing in an area immediately adjacent to the supraoptic nucleus but not within it. In contrast, labelling within the nucleus was found following anterograde transport of tracer deposited in the A1 region and in an area that includes the nucleus of the solitary tract. Neurones located in the perinuclear area were densely labelled by small injections into the supraoptic nucleus; they may represent a relay station for some afferent inputs to the supraoptic nucleus. These results suggest that the supraoptic nucleus is influenced by the same brain areas which project to its companion within the magnocellular system, the paraventricular nucleus.

Electrophysiological characteristics of immunochemically identified rat oxytocin and vasopressin neurones in vitro.

1. Intracellular recordings were made from supraoptic neurones in vitro from hypothalamic explants prepared from adult male rats. Neurones were injected with biotinylated markers, and of thirty‐nine labelled neurones, nineteen were identified immunocytochemically as containing oxytocin‐neurophysin and twenty as containing vasopressin‐neurophysin. 2. Vasopressin and oxytocin neurones did not differ in their resting membrane potential, input resistance, membrane time constant, action potential height from threshold, action potential width at half‐amplitude, and spike hyperpolarizing after‐potential amplitude. Both cell types exhibited spike broadening during brief, evoked spike trains (6‐8 spikes), but the degree of broadening was slightly greater for vasopressin neurones. When hyperpolarized below ‐75 mV, all but one neurone exhibited a transient outward rectification to depolarizing pulses, which delayed the occurrence of the first spike. 3. Both cell types exhibited a long after‐hyperpolarizing potential (AHP) following brief spike trains evoked either with a square wave pulse or using 5 ms pulses in a train. There were no significant differences between cell types in the size of the AHP evoked with nine spikes, or in the time constant of its decay. The maximal AHP evoked by a 180 ms pulse was elicited by an average of twelve to thirteen spikes, and neither the size of this maximal AHP nor its time constant of decay were different for the two cell types. 4. In most oxytocin and vasopressin neurones the AHP, and concomitantly spike frequency adaptation, were markedly reduced by the bee venom apamin and by d‐tubocurarine, known blockers of a Ca(2+)‐mediated K+ conductance. However, a minority of neurones, of both cell types, were relatively resistant to both agents. 5. In untreated neurones, 55% of vasopressin neurones and 32% of oxytocin neurones exhibited a depolarizing after‐potential (DAP) after individual spikes or, more commonly, after brief trains of spikes evoked with current pulses. For each neurone with a DAP, bursts of spikes could be evoked if the membrane potential was sufficiently depolarized such that the DAP reached spike threshold. In four out of five vasopressin neurones a DAP became evident only after pharmacological blockade of the AHP, whereas in six oxytocin neurones tested no such masking was found. 6. The firing patterns of neurones were examined at rest and after varying the membrane potential with continuous current injection. No identifying pattern was strictly associated with either cell type, and a substantial number of neurones were silent at rest.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunocytochemical, Golgi and electron microscopic characterization of putative dendrites in the ventral glial lamina of the rat supraoptic nucleus

Processes of magnocellular neurosecretory cells in the rat supraoptic nucleus which project along the pial surface in the ventral glial lamina were investigated using immunocytochemistry, Golgi stains and electron microscopy. Immunocytochemical studies revealed that although both oxytocin- and vasopressin-containing processes were evident in the ventral glial lamina, vasopressin-containing processes predominated. Ventral processes were thicker and of a different morphology than dorsal axon-like processes which joined the hypothalamo-neurohypophysial tract and exhibited large varicosities along their length or at their apparent termination. Golgi stains revealed that classically defined dendrites of supraoptic neurons projected primarily ventrally and often invaded the ventral glial lamina. No axons were traced to the lamina. Ultrastructurally, processes within the ventral glial lamina characterized as dendrites could be stained immunocytochemically for neurophysin and were post-synaptic to a variety of presynaptic elements. The results suggest that many dendrites from magnocellular neurosecretory cells in the supraoptic nucleus project to the ventral glial lamina and form a restricted, receptive plexus. The previously demonstrated coexistence of catecholamine-containing varicosities and other axon types with these processes in the lamina indicates an important role for supraoptic dendrites in integrating a wide variety of information relevant to neurohypophysial hormone release.

Stimulatory action of oxytocin on neurones of the dorsal motor nucleus of the vagus nerve

Extracellular recordings were obtained from spontaneously active neurones located in the dorsal motor nucleus of vagus nerve ( DMX ) in slices of the rat brainstem. Oxytocin applied to the bath at concentrations of 10(-7) M or 10(-6) M excited 79% of these cells in a concentration-dependent, reversible manner. The remaining cells were unaffected. The stimulatory effect of oxytocin was reversibly antagonized by a synthetic structural analogue known to block the peripheral, endocrine effects of neurohypophysial peptides. A selective oxytocic agonist was as potent as oxytocin, whereas vasopressin exerted a much weaker effect. We therefore suggest that neurones located in DMX are endowed with receptors for oxytocin.

Electrophysiological differences between oxytocin and vasopressin neurones recorded from female rats in vitro.

1. Intracellular recordings in vitro from immunochemically identified oxytocin (OT) and vasopressin (VP) neurones in the supraoptic nucleus (SON) of virgin or lactating female rats revealed no differences between neurone types in membrane potential (Vm), input resistance and current‐voltage relationships (I‐V), when taken at resting membrane potentials. 2. When OT (94%), but not VP, neurones (93%) were current clamped at depolarized voltages (above ‐50 mV), small hyperpolarizing pulses revealed a time‐ and voltage‐dependent outward rectification that was present above ‐75 mV and that decreased in amplitude as Vm approached the equilibrium potential for potassium (EK). The rectification was more pronounced when the neurones were held at a more depolarized membrane potential, and was larger the longer the neurone was held depolarized, reaching a maximum at 0.6‐0.9 s. 3. A rebound depolarization followed the offset of hyperpolarizing pulses that were associated with the rectification. The peak amplitude of the rebound showed a time and a voltage dependence. It followed a bell‐shaped curve as the hyperpolarizing commands were made larger, attaining a peak at ‐65 +/‐ 1.5 mV. The rebound amplitude increased with pulse duration, achieving a half‐maximal amplitude at 0.5 +/‐ 0.1 s. 4. The expression of the sustained outward rectification and the rebound in OT neurones was similar in virgin and lactating female rats. 5. These results indicate the presence of significant differences in the intrinsic membrane properties, probably K+ currents, between OT and VP neurones in both lactating and virgin female rats.

Spontaneous and osmotically-stimulated activity in slices of rat hypothalamus

Single unit activity was recorded from 400-500 mu m thick slices of rat hypothalamus, using either NaCl- or horseradish peroxidase-filled glass micropipettes. Spontaneous activity was present in the following hypothalamic loci: anterior hypothalamic-preoptic area, nucleus circularis, nucleus of the diagonal band of Broca, paraventricular accessory nucleus, paraventricular nucleus (all portions), periventricular regions of the anterior hypothalamus, and the suprachiasmatic nucleus. The supraoptic nucleus was the only major cell group studied to exhibit no spontaneous activity. Cells of the paraventricular and circularis nuclei were spontaneously active, displayed firing rates and patterns of activity similar to those recorded in vivo for magnocellular elements of the hypothalamus, and in some cases responded to increases in the osmolality of the bathing medium with altered firing rates and/or patterns of activity. Many cells in these preparations were characterized by phasic, bursting patterns of activity. Slow, irregular and regular, continuous activity was also frequently observed, as is typical in vivo. Median firing rates were in the range of 4-6 spikes/sec, somewhat faster than the rates usually reported for anesthetized in vivo preparations. These rates are more similar to those observed in unanesthetized monkeys or rats with diencephalic islands. Extracellular HRP marking provided a high degree of localization for many of the recorded cells. These results indicate that the hypothalamic slice preparation is useful for studies in which it is desirable to eliminate extrahypothalamic connections and in which it is necessary to exercise a fine degree of control over the extracellular environment of the cells.