Attenuation of induced-anxiety in rats by chlordiazepoxide: Role of raphe dorsalis benzodiazepine binding sites and serotoninergic neurons

In chronically implanted awake rats, microinjections of chlordiazepoxide (5 × 10^?7M) into the dorsal raphésignificantly attenuated the inhibition of lever-pressing for food elicited by a signal of punishment. This effect is abolished by prior application of 5,7-dihydroxytryptamine into the dorsal raphé(3 weeks after the infusion of the neurotoxin, dorsal raphétryptophan hydroxylase activity was reduced to 25% of control values). Furthermore, the disinhibitory effect of intra raphéchlordiazepoxide can be mimicked or potentiated by intra raphédorsalis application of serotonin (10^?7 or 10^?8 M, respectively). Further evidence for a crucial interaction between benzodiazepines and serotoninergic processes are provided by in vitro experiments showing that chlordiazepoxide or diazepam (10^?5 M) are able to facilitate the K⁺ -evoked [³H]serotonin release from rat midbrain slices. Finally, a high density of [³H]flunitrazepam binding sites was found in the dorsal (and the median) raphénucleus, the Kd and B_max values being not altered by prior infusion of 5,7-dihydroxytryptamine.These in vitro data suggest possible means by which intra raphé(and perhaps peripherally administered) benzodiazepines may affect the activity of serotoninergic neurons and thereby produce their effects on experimental anxiety.     

Transganglionic and anterograde transport of horseradish peroxidase across dorsal root ganglia: a tetramethylbenzidine method for tracing central sensory connections of muscles and peripheral nerves.

Horseradish peroxidase was applied either to severed sciatic nerves or to individual muscle groups in the lower leg of the rat. This horseradish peroxidase was taken up by sensory nerves and transported across the dorsal root ganglion and then anterogradely as far as central sensory terminals in the spinal cord and medulla. The dorsal root, dorsal column and the tract of Lissauer contained horseradish peroxidase labeled fibers. Following horseradish peroxidase application to the cut sciatic nerve, extraperikaryal reaction-product within the central processes of the sensory neurons was seen in the upper dorsal horn (substantia gelatinosa), lower dorsal horn, intermediate gray, ventral horn and nucleus of Clarke of the ipsilateral spinal cord; in the contralateral dorsal and ventral horns, and in the ipsilateral nucleus gracilis. Following intramuscular injections of horseradish peroxidase, the reaction-product was virtually absent in the upper dorsal horn and was concentrated in the lower dorsal horn, intermediate gray and ventral horn.These experiments show that horseradish peroxidase is transported anterogradely in significant quantities along the intact central processes of the dorsal root ganglion so that sensory terminations belonging to individual peripheral nerves or muscle groups can be traced. The demonstration of this complex transport was made possible with a sensitive histochemical method that used tetramethylbenzidine as the chromogen. Alternate sections processed with a commonly employed diaminobenzidine procedure did not show any of these central sensory connections.     

Serotonergic terminals in the neural sheath of the blowfly nervous system: electron microscopical immunocytochemistry and 5,7-dihydroxytryptamine labelling

With serotonin immunocytochemistry we have demonstrated an extensive plexus of immunoreactive varicose fibres in the neural sheath of the nervous system of the blowfly, Calliphora. These fibres are located in the neural sheath of the following regions: the maxillary-labial and labrofrontal nerves of the cerebral ganglia, the cervical connective, the dorsal surface of the thoracicoabdominal ganglia, two pairs of prothoracic nerves and the median abdominal nerve. We identified the serotonin-immunoreactive neural processes in the electron microscope by means of the peroxidase-antiperoxidase method. Immunoreactivity was seen in large granular vesicles (ca 100 nm), on membranes of smaller (ca 60 nm) and larger (ca 100 nm) agranular vesicles, along the inner surface of the axolemma, along neurotubules and outer membranes of mitochondria. By conventional electron microscopy we found numerous varicose neural processes in the neural sheath of some of the above regions. These varicosities are of at least two types. One type corresponds to the serotonin-immunoreactive profiles. A second type contains large granular vesicles (ca 200 nm) of variable electron density. 5,7-Dihydroxytryptamine injected into the head capsule labelled varicosities in the neural sheath, corresponding to the ones identified with serotonin immunocytochemistry. The electron-dense labelling was seen in flattened vesicles within these varicosities. We propose that the serotonin-immunoreactive fibers in the neural sheath constitute neurohemal regions for the release of serotonin into the circulation. The finding of another morphological type of varicose fibers in the neural sheath suggests the presence of further putative neurohormones in these regions.     

Opioid neurons and pain modulation: an ultrastructural analysis of enkephalin in cat superficial dorsal horn

To clarify the circuitry through which opioid compounds modulate spinal and trigeminal nociceptive transmission, we have examined the synaptic associations formed by leucine-enkephalin-containing (enkephalin) neurons in the superficial dorsal horn of the cat. As described previously, punctate enkephalin immunoreactivity is concentrated in the marginal layer (lamina I) and in both the outer and inner layers of the substantia gelatinosa (lamina IIo and IIi). In colchicine treated cats, enkephalin perikarya are most numerous in lamina I and at the border between laminae I and II. Ultrastructural analysis reveals that enkephalin cells receive a diverse afferent input. The majority of afferent inputs are presynaptic to the enkephalin dendrites; few axosomatic synapses are seen. Among these presynaptic axonal profiles are unlabeled axons which resemble primary afferent terminals, including the characteristic central axonal varicosity. Enkephalin dendrites are also postsynaptic to enkephalin immunoreactive axons. Two types of enkephalin axonal profiles appear in the superficial dorsal horn. Class I profiles are only found in lamina I. These are large profiles which form few synapses; those synapses made are axodendritic. Class II enkephalin axons are smaller and are distributed in both layers I and II. While Class II axons most commonly form axo-dendritic synapses, they also form axo-axonic synapses with flat vesicle-containing profiles; the latter are generally presynaptic to the enkephalin terminals. Serial analysis further revealed that both the enkephalin and the flat vesicle-containing profile synapse onto a common dendrite. Although enkephalin axons frequently lie adjacent to round vesicle-containing profiles, anatomical evidence that opioid axons form synapses with this type of ending was not found. An additional type of enkephalin vesicle containing-profile is found in layer IIi; its morphological features do not clearly distinguish its axonal or dendritic origin. These endings are typically postsynaptic to unlabelled central endings, and provide minimal presynaptic input to other elements in the neuropil. Like some class II axons, these labelled profiles contain vesicles which cluster at the membrane immediately adjacent to unlabelled central axons. These results indicate that spinal enkephalin neurons receive a variety of synaptic inputs. These include inputs which may derive from primary afferent axons. Enkephalin neurons, in turn, influence nociceptive transmission predominantly through postsynaptic mechanisms. Finally, while we did not observe enkephalin terminals presynaptic in an axoaxonic relationship, the possibility that enkephalin neurons modulate the excitability of fine fiber nociceptive and nonnociceptive afferents via "nonsynaptic interactions" is discussed.     

Distribution of serotonin-immunoreactivity in the central nervous system of the rat—Cell bodies and terminals

The localization and distribution of serotonin (5-hydroxytryptamine, 5-HT) has been studied with the indirect immunofluorescence technique using a highly specific and well-characterized antibody to 5-HT. In neuron systems 5-HT was found to be primarily present with a distribution similar to that observed in basic mappings carried out with the formaldehyde-induced fluorescence method. In addition to the nine areas originally described, several other areas in the mesencephalon and rhombencephalon appeared to contain widely distributed 5-HT-positive perikarya. In the median eminence 5-HT fluorescent mast cells could be visualized. No 5-HT-positive nerve cell bodies could be observed either in the telencephalon or diencephalon.Our results also demonstrate a widespread occurrence of 5-HT-positive nerve terminals throughout the central nervous system. Dense populations of serotonin-immunoreactive nerve terminals are present in the following areas, from rostral to caudal: various parts of the medial forebrain bundle; the ventromedial part of the nucleus suprachiasmaticus: the most ventrolateral part of the caudatus-putamen complex; an area between the rostral part of the nucleus reuniens and the fornix: at that same level of the latter area, a region just ventral of the fornix; the dorsolateral part of the nucleus periventricularis thalami; an area lateral of the claustrum, close to the cortex piriformis; the nucleus amygdaloideus basalis; the dorsomedial part of the nucleus medialis thalami pars medialis, only in the rostral part; the inner part of the nucleus ventromedialis hypothalami; the ventral part of the nucleus mamillaris medialis; the radiatio thalami superior; the nucleus subthalamicus; the caudal part of the nucleus amygdaloideus medialis posterior; the area pretectalis radialis; the dorsal part of the nucleus ventralis corporis geniculati lateralis; the ventromedial part of the substantia nigra reticularis; an area ventral of the decussatio supramammillaris; the substantia nigra pars lateralis; the dorsolateral part of the nucleus reticularis pontis oralis; some parts of the nucleus origines nervi trigemini; the ventromedial part of the nucleus origines nervi facialis; the medial part of the nucleus tractus solitarius and the medial part of the cornu ventrale of the spinal cord. There are only a few areas where no serotonin-immunoreactivity could be found. Within several areas, as, e.g., hippocampus, nucleus caudatus, medial forebrain bundle, the density of serotoninergic fibers appeared to show considerable local differences. Within the same nuclei or area the amount can differ considerably.This immunohistochemical procedure makes it possible to study the distribution of serotonin-containing nerves and their processes in considerable detail. The finding that serotonin-containing neurons are present in many nuclei that also include catecholamine-containing neurons makes it necessary to reconsider the terminology of the monoaminergic cell groups.     

Uptake and retention of [3H]adrenaline by central monoaminergic neurons: a light- and electron-microscope radioautographic study after intraventricular administration in the rat

Paraventricular and paracisternal regions of adult rat central nervous system were investigated by light- and electron-microscope radioautography after intraventricular administration of tritiated adrenaline. In tissue primarily fixed by glutaraldehyde perfusion and post-fixed by immersion in osmium tetroxide, there were no aggregates of silver grains indicative of intraneuronal accumulation of the tracer, except over perivascular nerve terminals at the base of the brain. In contrast, when both fixation and postfixation were carried out by rapid vascular perfusion, preferentially labeled nerve cell bodies and axonal varicosities (i.e. terminals) were detected in various anatomical areas known to contain dopaminergic and/or noradrenergic neurons. Serotoninergic axonal varicosities in the supraependymal plexus and subcommissural organ, as well as a small group of nerve cell bodies of undetermined chemical identity in the n. paraventricularis thalami were also found to be labeled. Addition of a ten-fold higher concentration of non-radioactive serotonin to the solution of [3H]adrenaline suppressed the reactivity in the subcommissural organ and the supraependymal plexus but had no such effect elsewhere in brain. Lesioning of the nigrostriatal dopaminergic system with 6-hydroxydopamine prior to [3H]adrenaline injection eradicated axon terminal labeling in the ipsilateral neostriatum. Electron-microscopic examination of [3H]adrenaline-labeled varicosities in the neostriatum, lateral septum, arcuate nucleus and median eminence extended earlier observations on the ultrastructure of the catecholaminergic innervation of these regions. It was concluded that both dopaminergic and noradrenergic neurons as well as certain serotonin-containing axon terminals can take up and retain [3H]adrenaline, although they probably have lesser affinity for this amine than for their own transmitter. Due to the fact that presumptive adrenergic neurons are intermingled with dopaminergic and noradrenergic elements, further work will be needed to determine to which extent they also contributed to [3H]adrenaline uptake in the present experimental conditions.     

Actions of opiates, substance P, and serotonin on the excitability of primary afferent terminals and observations on interneuronal activity in the neonatal rat's dorsal horn in vitro

Approximately 5 segments of lumbo-thoracic spinal cord together with connected dorsal root ganglia were removed from 1-11-day-old rats and maintained in vitro. Dorsal root afferents, recorded from the ganglion and stimulated at the root entry zone, had conduction velocities typical of unmyelinated fibers (less than 2 m/s). The spinal terminals of individual afferents showed increased excitability with bath application of substance P and serotonin and decreased excitability with morphine sulfate, [D-ala2]methionine-enkephalinamide, manganese ions and magnesium ions. Naloxone by itself elicited no change in excitability, although it appeared to reduce the ongoing effect of opiates. Neurons recorded extracellularly in the dorsal horn responded to afferent volleys with one or more of 3 distinct phases: an excitation roughly coincident with the volley's arrival, a 50-300 ms period of inhibition, and a late excitation of 150-300 ms latency. The excitability results are accounted for by a model in which substance P, gamma-aminobutyric acid and possibly other depolarizing agents are contained in interneurons which synapse on afferent terminals. These interneurons could receive inhibitory enkephalinergic input, and, in the neonate but not the adult, excitatory serotoninergic input. An alternate scheme would have enkephalin and serotonin acting directly on afferent terminals, although perhaps by non-synaptic diffusion since the appropriate synapses have not been seen in histochemical studies. Such an action for enkephalin might explain the existence of opiate receptor on afferent terminals. The interneuronal responses to afferent volleys are parallel in most aspects to those found in the dorsal horns of adult mammals in vivo.     

Distribution of serotonin in the brain stem and spinal cord of the lizard Varanus exanthematicus: an immunohistochemical study

The distribution of serotonin-containing nerve cell bodies, fibers and terminals in the lizard Varanus exanthematicus was studied with the indirect immunofluorescence technique, using antibodies to serotonin. Most of the serotonin-containing cell bodies were found in the midline, in both of the raphe nuclei, i.e. the nuclei raphes superior and inferior. A considerable number of more laterally shifted serotonergic neurons was found particularly at three levels of the brain stem, viz. in the caudal mesencephalic tegmentum, at the isthmic level, and over a long distance in the medulla oblongata. These laterally situated serotonin-positive neurons were partly found within the confines of the substantia nigra, the nucleus reticularis superior and the lateral part of the nucleus reticularis medius and ventrolateral part of the nucleus reticularis inferior, respectively. No serotonergic cell bodies were found in the spinal cord. In the brain stem a dense serotonergic innervation was observed in all of the motor nuclei of the cranial nerves, in two layers of the tectum mesencephali, in the nucleus interpeduncularis pars ventralis, the nucleus profundus mesencephali pars rostralis, the periventricular grey, the nucleus parabrachialis, the vestibular nuclear complex, the nucleus descendens nervi trigemini, the nucleus raphes inferior, and parts of the nucleus tractus solitarii. Descending serotonergic pathways could be traced into the spinal cord via the dorsolateral, ventral and ventromedial funiculi, and were found to innervate mainly three parts of the spinal grey throughout the spinal cord, i.e. the dorsal part of the dorsal horn, the motoneuron area in the ventral horn, and the intermediate zone just lateral to the central canal. The results obtained in the present study suggest a close resemblance of the organization of the serotonergic system in reptiles and mammals, especially as to the serotonergic innervation of the spinal cord.     

Simultaneous determination of indole- and catecholamines in small brain regions in the rat using a weak cation exchange resin

A fluorometric procedure is described which allows the measurement of nanogram amounts of serotonin, horepinephrine, and dopamine in small brain areas (20–350 mg) from individual rats. The amines are separated from their precursor amino acids and acid metabolites by a single-column chromotography step using a weak cation exchange resin. It is possible quantitatively to determine 10–30 ng of each amine when all three are measured simultaneously. When either serotonin or the catecholamines are assayed in a tissue sample, as little as 5–15 ng may be detected. Recoveries of the amines range from 85 to 93% as measured by addition of¹⁴C-labeled amines to tissue supernatants.     

Cortical relay neurons and interneurons in the N. ventralis posterolateralis of cats: a horseradish peroxidase, electron-microscopic, Golgi and immunocytochemical study

After injections of horseradish peroxidase involving the whole primary (SI) and secondary somatosensory (SII) areas of adult cats, 16-21% out of 2220 counted neurons in the nucleus ventralis posterolateralis were unlabelled. The mean areas of perikarya of these neurons varied between 111.8 +/- 32.3 microns2 and 180.8 +/- 48.6 microns2. The size of perikarya of retrogradely-labelled neuron ranged from 256.9 +/- 100.4 microns2 to 409 +/- 163 microns2. Retrogradely-labelled and unlabelled neurons were examined under light- and high-voltage electron-microscopy. Besides 'large', mainly multipolar or oval fusiform perikarya, retrogradely-labelled neurons may display perikarya of 'small' size. Both types of neurons correlate well with Golgi-impregnated cells with a tufted dendritic pattern usually identified as thalamocortical neurons. On the other hand, the size and morphology of perikarya and initial dendrites of neurons unlabelled by retrograde transport of horseradish peroxidase correlate well with that of Golgi-impregnated neurons which are markedly different from the thalamocortical neurons, have very characteristic and profuse dendritic appendages and have been identified by previous investigators as Golgi Type II neurons. In order to probe further whether these may correspond to the GABAergic interneurons proposed by previous evidence, an immunocytochemical approach was also applied at the light- and electron-microscope level, using an antiserum prepared in sheep against rat brain glutamate decarboxylase. By this method it is shown that 19-21% of neurons in the nucleus ventralis posterolateralis of adult cats are glutamate decarboxylase-positive and that the perikaryal size of these labelled neurons ranges between 134.6 +/- 44.5 microns2 and 164.4 +/- 47.3 microns2. Histogram distribution of the number and areas of the counted immunoreactive neurons closely matches that of unlabelled neurons in experiments with retrograde transport of horseradish peroxidase. The results give support to previous evidence suggesting that part of population of neurons in the nucleus ventralis posterolateralis is represented by a distinct class of neurons which are apparently GABAergic.     

Light- and electron-microscopic immunocytochemistry of glutamic acid decarboxylase (GAD) in the basal hypothalamus: Morphological evidence for neuroendocrine γ -aminobutyrate (GABA)

GABAergic cells and axon terminals were localized in the basal hypothalamus of different species (rat, mouse and cat), by means of an immunocytochemical approach using a specific and well-characterized antiserum to the GABA biosynthetic enzyme, glutamate decarboxylase. Lightmicroscopic visualization was performed with an indirect immunofluorescence method and electron-microscopic observations were made on material with pre-embedding staining and use of the peroxidase-antiperoxidase procedure.At the light-microscopic level, a dense immunofluorescent plexus was observed over both the medial and lateral parts of the external layer of the median eminence. The labelling extended from the rostral part of the median eminence up to the pituitary stalk. Over the subependymal and internal layers only a few immunoreactive dots were visible, except around the blood vessels where they appeared more concentrated. Immunoreactive varicosities could be found following the outlines of the capillary loops and lining tanycyte processes, especially in the median eminance midportion.At the electron-microscopic level, the immunolabelling was exclusively found over neuronal profiles in the median eminence. The latter represented a small fraction of the total number of varicosities visible on the same section. Labelled profiles typically contained numerous small clear synaptic vesicles and only a few or no dense-core vesicles. In the subependymal and internal layers, rare labelled endings were found close to ependymal cells or among transversally cut fibers, respectively. In the palisadic zone, elongated positive boutons were visible intermingled with bundles of unlabelled axons and glial or ependymal processes. In the neurohemal contact zone, immunoreactive endings were observed among unlabelled neurosecretory endings in close vicinity to fenestrated capillary perivascular space.Small moderately intense immunofluorescent varicosities were observed all over the hypothalamus. The density of the glutamate decarboxylase-positive network was higher than in most diencephalic regions. Intraventricular or topical injection of colchicine allowed the visualization of small lightly immunoreactive cells in the diffusion area of colchicine. In the arcuate nucleus labelled axonal endings containing small pleomorphic synaptic vesicles and sometimes a few dense-core vesicles were observed at the electron-microscopic level. Typical synaptic junctions were commonly found between positive endings and unlabelled perikarya, or more frequently, unlabelled dendrites.These findings show that glutamate decarboxylase-containing endings are localized in several strategic sites for potential GABAergic neuroendocrine regulations. The GABAergic endings found among neurosecretory endings in the neurohemal contact zone may provide the morphological support for the release of γ -aminobutyrate into the portal blood flow as an hypothalamic hypophysiotropic hormone. Alternatively, neurosecretory cells might be under GABAergic control expressed either at their terminal level within the median eminence or the cell body level within the parvicellular hypothalamic nuclei.     

Morphological and physiological properties of neostriatal neurons: An intracellular horseradish peroxidase study in the rat

The physiological and morphological (light and electron microscopic) properties of four categories of neostriatal neurons (two types of medium spiny cells and two types of aspiny cells) were analyzed using the technique of intracellular recording and intracellular labeling with horseradish peroxidase. All of the neurons in this study had excitatory responses following stimulation of the cortex and substantia nigra except for the large aspiny neuron for which only substantia nigra inputs were tested. Morphologically, these neurons differed with respect to the size and shape of their somata, density and distribution of dendritic spines and distribution of their axons and axon collaterals. Ultrastructurally, observed somatic differences included the quantity and distribution of organelles and conformation of the nuclear envelope.The axons of one type of medium spiny neuron and the large aspiny neuron were myelinated. Unmyelinated axon collaterals arose from the axons of both types of medium spiny neurons and formed synapses on the dendritic shafts and possibly with the necks of spines of other neostriatal neurons. The parent axons of the most common type of medium spiny neurons were followed to the globus pallidus and, in some cases, to the internal capsule.     

The differential distribution and relationship of serotoninergic and peptidergic fibers to sympathoadrenal neurons in the intermediolateral cell column of the rat: a combined retrograde axonal transport and immunofluorescence study

The preganglionic sympathetic neurons in the intermediolateral cell column of the thoracic and upper lumbar segments of the spinal cord which innervate the chromaffin cells in the adrenal medulla, sympathoadrenal preganglionic neurons, were identified by the method of retrograde axonal transport of the fluorescent dyes Fast Blue and True Blue. In rats, Fast Blue or True Blue was injected into the medulla of the left adrenal gland. After a survival period of 5 days, the animals were perfusion fixed, the thoracic and lumbar spinal cord sectioned and processed for the immunofluorescent localization of met-enkephalin, neurophysin, oxytocin, serotonin, somatostatin and substance P immunoreactivity. Neuronal perikarya which were retrogradedly-labeled with Fast Blue or True Blue were observed in the intermediolateral cell column from the T1 to the L2 spinal cord segments. The distribution of the sympathoadrenal neurons was determined by counting the number of retrogradedly-labeled neurons per spinal cord segment. In the five animals used for quantifying the sympathoadrenal preganglionic neurons, the majority (72.3%) of the retrogradely-labeled neurons counted per spinal cord were located within the T7-T12 segments. The T9 segment contained the largest average number (20.1%) of retrogradely-labeled cells in a single segment. Met-enkephalin, serotonin and substance P immunoreactive fibers were prominent in the intermediolateral cell column, whereas oxytocin, neurophysin and somatostatin immunoreactive fibers were sparse. The met-enkephalin, serotonin and substance P fibers were seen surrounding both unlabeled and retrogradely-labeled neurons; somatostatin fibers appeared to preferentially contact retrogradely-labeled neurons; whereas, the neurophysin and oxytocin fibers were not found in proximity to retrogradely-labeled neurons. Met-enkephalin, neurophysin, oxytocin, somatostatin and substance P immunoreactivity were depleted in the intermediolateral cell column below the level of a spinal cord transection. Serotonin immunoreactivity was depleted in the intermediolateral cell column below the level of the transection for five to six segments, but sparse networks of immunoreactive fibers were observed in both the intermediolateral cell column and the ventral horn in more caudal segments. Met-enkephalin, serotonin, somatostatin and substance P immunoreactivity were decreased in both the contralateral and ipsilateral intermediolateral cell column below the level of a spinal cord hemisection, suggesting that both crossed and uncrossed descending pathways exist. Neurophysin and oxytocin immunoreactivity were depleted below the level of the hemisection in the ipsilateral intermediolateral cell column without noticeable decrease in the level of immunoreactivity in the contralateral intermediolateral cell column, suggesting that a decussation does not occur at the level of the spinal cord, but may exist above the level of the hemisection...     

The role of a raphe serotonin system in the control of septal unit activity and hippocampal desynchronization.

The effects of electrical stimulation of the median raphe nucleus on neuronal discharges in the medial septal area and electrical activity of the hippocampus were examined in urethane-anaesthetized rats. Two cell populations in the medial septal area were differentiated on the basis of their spontaneous discharge pattern, response to median raphe nucleus stimulation and whether or not they were antidromically activated following hippocampal stimulation. Medial septal area cells classified as I-neurones discharge in an irregular pattern which was unrelated to hippocampal activity. In contrast, B-neurones discharge in either rhythmic bursts or in an irregular manner which were related to hippocampal ‘theta’ or ‘desynchronization’, respectively. Single pulse stimulation of the median raphe nucleus inhibited the spontaneous discharge of I-neurones but did not influence the firing of B-neurones or hippocampal activity. Repetitive stimulation of the median raphe nucleus resulted in a prolonged inhibition of I-neurones, a disruption of the bursting discharge of B-neurones, and desynchronization of the hippocampal ‘theta’. The effects of median raphe nucleus stimulation were blocked by pretreatment with parachlorophenylalanine suggesting that a serotonin-containing system originating in the median raphe nucleus may be involved in mediating these responses.     

Study of the efferent connections of the enkephalinergic magnocellular dorsal nucleus in the guinea-pig hypothalamus using lesions, retrograde tracing and immunohistochemistry: evidence for a projection to the lateral septum

The efferents of enkephalin-immunoreactive neurons in the magnocellular dorsal nucleus of the guinea-pig were studied using different neuroanatomical methods and indirect immunocytochemical technique. Following unilateral implantation of the fluorescent dye 4',6-diamidino-2-phenylindole in the lateral septal nucleus, retrogradely-labeled perikarya were found in the magnocellular dorsal nucleus. These labeled perikarya reacted with antiserum against enkephalin, demonstrating that enkephalin-immunoreactive neurons in the magnocellular dorsal nucleus project to the lateral septal nucleus. In other experiments, complete bilateral lesions were produced in the magnocellular dorsal nucleus by electrocoagulation. Enkephalin-immunoreactive nerve fibers and terminals were totally depleted in the lateral septal nucleus. This confirms that septal enkephalin-immunoreactive terminals originate in the magnocellular dorsal nucleus and further suggests that this nucleus is the source of all the enkephalin-immunoreactive material found in the septum. Experiments utilizing two different fluorescent dyes, 4',6-diamidino-2-phenylindole and propidium iodide, injected in each side of the lateral septal nucleus, respectively, demonstrated that the magnocellular dorsal nucleus gives off axon collaterals to both sides of the septum, since double-labeling of individual cell bodies was detected in the nucleus. By relating this finding to the results obtained after unilateral destruction of the nucleus, which caused an incomplete loss of enkephalin- immunoreactive material in the lateral septal nucleus ipsilaterally, it is suggested that the enkephalinergic hypothalamo-septal pathway contains unbranching neurons projecting ipsilaterally and branching neurons distributing fibers ipsilaterally and contralaterally. Lesion experiments, and experiments based on the retrograde axonal transport of horseradish peroxidase after intravenous injections, demonstrated that the magnocellular dorsal nucleus contributes neither to the tubero-infundibular nor to the hypothalamo-neurohypophyseal tracts. The lateral septal nucleus receives numerous aminergic and peptidergic projections, indicating the potential importance of this region in physiological and behavioral events. In the guinea-pig, the well-demarcated enkephalinergic pathway demonstrated in this study provides a convenient model for the experimental study of the enkephalinergic innervation of the lateral septal nucleus.     

The morphology of neurons in trigeminal nucleus oralis projecting to the medullary dorsal horn (trigeminal nucleus caudalis): a retrograde horseradish peroxidase and Golgi study

This study demonstrates that trigeminal nucleus oralis, the most rostral subdivision of the spinal trigeminal nucleus, contains four morphologically distinct types of small neurons which project to the medullary dorsal horn (trigeminal nucleus caudalis) via descending intratrigeminal pathways. Using the retrograde transport of horseradish peroxidase following injections in the medullary dorsal horn, labeled small neurons with cell bodies ranging from 8-15 microns in diameter are found principally in the ventrolateral portion of the trigeminal nucleus oralis. Most neurons are labeled ipsilaterally throughout the entire rostrocaudal extent of the ventrolateral portion of the trigeminal nucleus oralis, but a few cells are also labeled contralaterally. From this aspect of the present study it can be concluded that a specific portion of the trigeminal nucleus oralis, i.e. the ventrolateral part, contains numerous small neurons which send descending projections to the medullary dorsal horn that could affect synaptic activity there. Utilizing both the methods of Golgi and retrograde horseradish peroxidase labeling four distinct types of small descending medullary dorsal horn projection neurons can be distinguished in the ventrolateral portion of the trigeminal nucleus oralis on the basis of their morphology and the distribution of their axons and dendrites. All four neuronal cell types are present throughout the entire rostrocaudal extent of the trigeminal nucleus oralis. Type I neurons are the most frequently labeled descending medullary dorsal horn projection neurons. They are concentrated in the medial 500-550 microns of the ventrolateral portion of the trigeminal nucleus oralis and display dendritic trees which occupy spherical domains approaching 300 microns in diameter. The unmyelinated axons of many of these cells arise either directly from the cell body or a primary dendrite and give rise to a single collateral within 50 microns of their site of origin. This collateral generates a fine axonal plexus within a portion of the dendritic arbor of the parent cell while the parent axon, without branching further, travels a short distance in the ventrolateral portion of the trigeminal nucleus oralis and enters a deep axon bundle. Type II neurons are the second most frequently labeled descending medullary dorsal horn projection neuron. They generate medial and lateral dendritic arbors which together span nearly the entire medial 500-550 microns of the ventrolateral portion of the trigeminal nucleus oralis. An unmyelinated axon emerges from the cell body and within 10-30 microns of its origin gives rise to two collaterals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cerebrovascular serotonergic receptors mediating vasoconstriction: further evidence for the existence of 5-HT2 receptors in rat and 5-HTVIike receptors in guinea-pig basilar arteries

Pharmacological experiments were carried out on isolated basilar arteries (BA) from the brain vasculature of guinea-pig and rat in order to characterize post-junctional serotonergic receptors mediating contraction by the use of selective agonists and antagonists. The sensitivity to 5-HT was higher, but the intrinsic activity lower, in guinea-pig compared to rat vessels. The contractile potency of the 5-HT₁ agonist, 5-carboxamidotryptamine (5-CT), was three times higher than 5-HT in guinea-pig but 16 times lower in rat BA. In arteries from both species the 5-HT_1A agonist, 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), only caused weak contraction. In rat BA, where the serotonergic contractile receptors are ketanserin-sensitive, mesulergine inhibited the contraction in doses high enough to block 5-HT₂ receptors, and also propranolol slightly inhibited the contraction, probably due to its binding to these receptors. Methiothepin, a potent antagonist of the 5-HT₁-like receptors, affected the contraction in a non-competitive manner. The antagonist profile was different in guinea-pig BA: propranolol was ineffective, mesulergine caused a slight, non-surmountable inhibition, whereas methiothepin acted as a true, competitive antagonist. The data support previous suggestions that the serotonergic contraction in rat BA is mediated by 5-HT₂ receptors, whereas the present data show that 5-HT₁-like receptors predominate in guinea-pig BA.     

Different neuronal localization of aspartate-like and glutamate-like immunoreactivities in the hippocampus of rat, guinea-pig and Senegalese baboon (Papio papio), with a note on the distribution of gamma-aminobutyrate

Antisera were raised in rabbits against aspartate or glutamate conjugated to bovine serum albumin by glutaraldehyde. After immunosorbent purification the antisera reacted selectively with brain protein-glutaraldehyde conjugates of the respective amino acids. These results from model systems encouraged us to employ the antisera to study the distribution of free aspartate and glutamate in brain tissue. The aspartate antiserum produced intense staining of interneurons and deep hilar neurons and modest labelling of pyramidal and granular cells in the hippocampal formation of rats, guinea-pigs and baboons perfusion-fixed with glutaraldehyde. In contrast, glutamate-like immunoreactivity was generally high in pyramidal and granular cells and low in interneurons. In hippocampal slices immersion fixed in glutaraldehyde after being soaked in Krebs' solution aspartate-like and glutamate-like immunoreactivities were lost from perikarya and dendrites. The staining that remained occurred in nerve terminal-like dots and matched the distribution of the major excitatory fiber systems, except that only glutamate-like immunoreactivity, and not aspartate-like immunoreactivity, was concentrated at the site of the mossy fiber terminals, and that aspartate-like but not glutamate-like immunoreactivity occurred between the granular and pyramidal cell bodies. The present technique specifically demonstrates aspartate and glutamate in glutaraldehyde-fixed tissue. We suggest that in perfusion-fixed material the staining intensities reflect the total concentrations of the amino acids (i.e. the "metabolic pool" plus the "transmitter pool"). In immersion-fixed hippocampal slices the "transmitter pool" may be preferentially visualized.