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Nucleus tractus solitarius and giant-cell and lateral reticular nuclei were studied using the reaction to NADPH-diaphorase in 7-, 10-, 14-, 30-, 45-, 60-day-and 3- and 6-month-old rats receiving L-NAME (50 μg/kg, 2 times a day) on days 1-6 of life. In 7-14-day-old rats, the compound reduced NO-synthase activity in the majority of NO-neurons and the total number and to a lesser degree the relative number of these neurons, while cell cross-section areas remained practically unchanged. The differences in the corresponding quantitative parameters between the control (D-NAME administration) and experimental groups decreased with time after the last L-NAME injection and became undetectable starting from the age of 30-45 days. In the nucleus tractus solitarius, the changes in metric parameters after exposure to NO-synthase inhibitor were more pronounced than in the reticular formation nuclei.

The central mesencephalic reticular formation (cMRF) likely plays a role in gaze control, as cMRF neurons receive tectal input and provide a bilateral projection back to the superior colliculus (SC). We examined the important question of whether this feedback is excitatory or inhibitory. Biotinylated dextran amine (BDA) was injected into the cMRF of M. fascicularis monkeys to anterogradely label reticulotectal terminals and retrogradely label tectoreticular neurons. BDA labeled profiles in the ipsi- and contralateral intermediate gray layer (SGI) were examined electron microscopically. Postembedding GABA immunochemistry was used to identify putative inhibitory profiles. Nearly all (94.7%) of the ipsilateral BDA labeled terminals were GABA positive, but profiles postsynaptic to these labeled terminals were exclusively GABA negative. In addition, BDA labeled terminals were observed to contact BDA labeled dendrites, indicating the presence of a monosynaptic feedback loop connecting the cMRF and ipsilateral SC. In contrast, within the contralateral SGI, half of the BDA labeled terminals were GABA positive, while more than a third were GABA negative. All the postsynaptic profiles were GABA negative. These results indicate the cMRF provides inhibitory feedback to the ipsilateral side of the SC, but it has more complex effects on the contralateral side. The ipsilateral projection may help tune the "winner-take-all" mechanism that produces a unified saccade signal, while the contralateral projections may contribute to the coordination of activity between the two colliculi.

The ventral part of the oral pontine reticular nucleus (vRPO) is involved in the generation and maintenance of rapid eye movement (REM) sleep. Both GABAergic and serotonergic neurotransmission have been implicated in the control of the sleep-wakefulness cycle. Nevertheless, the synaptic organization of serotonergic terminals in the vRPO has not yet been characterized. We performed an electron microscope study of serotonin-immunoreactive (5-HT-IR) terminals using immunoperoxidase or immunogold-silver methods. In a second set of experiments, combining GABA immunoperoxidase and 5-HT immunogold-silver techniques, we examined inputs from GABA-immunoreactive (GABA-IR) terminals to serotonergic neurons. 5-HT-IR terminals were located primarily on dendrites and occasionally on somata of unlabeled and 5-HT-IR neurons. The majority of the synapses formed by 5-HT-IR terminals were of the symmetrical type, making contacts primarily with unlabeled dendritic profiles. Moreover, 5-HT-IR terminals contacted unlabeled axon terminals that formed asymmetric synapses on dendrites. Double immunolabeling experiments showed 5-HT-IR and GABA-IR afferents, in apposition to each other, making synapses with the same dendrites. Finally, GABA-IR terminals innervated 5-HT-IR and GABA-IR dendrites. Our findings indicate that serotonin would modulate the neuronal activity through inhibitory or excitatory influences, although the action of serotonin on the vRPO would predominantly be inhibitory. Moreover, the present results suggest that the serotonin modulation of vRPO neurons might involve indirect connections. In addition, GABA might contribute to the induction and maintenance of REM sleep by inhibiting serotonergic and GABAergic neurons in the vRPO.

The rostral ventrolateral medulla (RVLM), a region critical for the tonic and reflex control of arterial pressure, contains a group of adrenergic (C1) neurons that project to the spinal cord and directly modulate pre-ganglionic sympathetic neurons. Epidemiological data suggest that there are gender differences in the regulation of blood pressure. One factor that could be involved is angiotensin II signaling and the associated production of reactive oxygen species (ROS) by NADPH oxidase, which is emerging as an important molecular substrate for central autonomic regulation and dysregulation. In this study dual electron microscopic immunolabeling was used to examine the subcellular distribution of the angiotensin type 1 (AT(1)) receptor and two NADPH oxidase subunits (p47 and p22) in C1 dendritic processes, in tissue from male, proestrus (high estrogen) and diestrus (low estrogen) female rats. Female dendrites displayed significantly more AT(1) labeling and significantly less p47 labeling than males. While elevations in AT(1) labeling primarily resulted from higher levels of receptor on the plasma membrane, p47 labeling was reduced both on the plasma membrane and in the cytoplasm. Across the estrous cycle, proestrus females displayed significantly higher levels of AT(1) labeling than diestrus females, which resulted exclusively from plasma membrane density differences. In contrast, p47 labeling did not change across the estrous cycle, indicating that ROS production might reflect AT(1) receptor membrane density. No significant differences in p22 labeling were observed. These findings demonstrate that both sex and hormonal levels can selectively affect the expression and subcellular distribution of components of the angiotensin II signaling pathway within C1 RVLM neurons. Such effects could reflect differences in the capacity for ROS production, potentially influencing short term excitability and long term gene expression in a cell group which is critically involved in blood pressure regulation, potentially contributing to gender differences in the risk of cardiovascular disease.

Synaptic vesicle recycling has been proposed to depend on proteins which coordinate membrane and cytoskeletal dynamics. Here, we examine the role of the dynamin- and N-WASP (neural Wiskott-Aldrich syndrome protein)-binding protein syndapin/PACSIN at the lamprey reticulospinal synapse. We find that presynaptic microinjection of syndapin antibodies inhibits vesicle recycling evoked by intense (5 Hz or more), but not by light (0.2 Hz) stimulation. This contrasts with the inhibition at light stimulation induced by perturbation of amphiphysin (Shupliakov et al., 1997). Inhibition by syndapin antibodies was associated with massive accumulation of membranous cisternae and invaginations around release sites, but not of coated pits at the plasma membrane. Cisternae contained vesicle membrane, as shown by vesicle-associated membrane protein 2 (VAMP2)/synaptobrevin 2 immunolabeling. Similar effects were observed when syndapin was perturbed before onset of massive endocytosis induced by preceding intense stimulation. Selective perturbation of the Src homology 3 domain interactions of syndapin was sufficient to induce vesicle depletion and accumulation of cisternae. Our data show an involvement of syndapin in synaptic vesicle recycling evoked by intense stimulation. We propose that syndapin is required to stabilize the plasma membrane and/or facilitate bulk endocytosis at high release rates.

Ultrastructural changes in the lateral reticular nuclei of the medulla oblongata of rat pups developing under the effect of chronic mental and pain stress indicate impaired histogenesis of structures of the medulla oblongata reticular formation and appearance of pronounced morphofunctional differences between the neurons.

Many biochemical, physiological and behavioural processes, from bacteria to human, exhibit roughly 24 h cyclic oscillations defined as circadian rhythms. However, during ageing, numerous aspects of the circadian biology undergo alterations; in particular, the sleep pattern changes, with more frequent awakenings and shorter sleep time. The basic mechanism of the circadian clock relies on intracellular molecular pathways involving interlocking transcriptional/translational feedback loops, and CLOCK protein, a transcription factor, is essential for normal circadian rhythms. In this study, the fine distribution of CLOCK protein has been analysed, in adult and old rats, at different phases of the daily cycle in the neurons of the medullary reticular formation, involved in the control of the sleep-wake cycle. The results demonstrate quali-quantitative modifications of CLOCK protein in the neurons of old animals, suggesting that such a deregulation of the intracellular clock mechanism may play some role in the degeneration of the sleep-wake circadian cycle.

Kappa opioid receptor (KOR) ligands alter nociceptive responses when applied to the rostral ventromedial medulla (RVM). However, the effects of kappa opioid receptor ligands are distinct in males and females. The present study examined the distribution of kappa opioid receptor immunoreactivity in the RVM of male and female rats. KOR immunoreactivity was found at pre- and postsynaptic sites within the RVM of both sexes. The most common KOR-immunoreactive (KOR-ir) neuronal structures were unmyelinated axons, followed by axon terminals, dendrites, and somata. Different proportions of KOR-ir axon terminals and dendrites were found in females at different estrous stages. Specifically, dendrites containing KOR immunoreactivity were less abundant in proestrus females compared with estrus females and showed a trend toward being less abundant in males, suggesting that KOR ligands applied to the RVM may be less potent in proestrus females. These findings suggest that the distribution of KORs in the RVM may be influenced by reproductive hormone levels. We also found KOR immunoreactivity in many spinally projecting neurons within the RVM of female rats. These findings are consistent with the hypothesis that KOR ligands influence nociceptive behaviors by altering the activity of specific populations of neurons within the RVM. The abundance of KOR in axons and axon terminals in RVM indicates a substantial role for presynaptic effects of KOR ligands through pathways that have not been clearly delineated. Altering the balance between pre- and postsynaptic receptive sites may underlie differences in the effects of KOR agonists on nociceptive responses in males and females., (Copyright 2006 Wiley-Liss, Inc.)

The ventral division of the reticular oral pontine nucleus (vRPO) is a pontine tegmentum region critically involved in REM sleep generation. Previous reports of morphine microinjections in the cat pontine tegmentum have shown that opioid receptor activation in this region modulates REM sleep. Even though opiate administration has marked effects on sleep-wake cycle architecture, the distribution of opioid receptors in vRPO has only been partially described. Using an antiserum directed against delta opioid receptor (DOR), to which morphine binds, in the present study, we use (1) light microscopy to determine DOR cellular distribution in the rostral pontine tegmentum and (2) electron microscopy to determine DOR subcellular distribution in the cat vRPO. In the dorsal pons, DOR immunoreactivity was evenly distributed throughout the neuropil of the reticular formation and was particularly intense in the parabrachial nuclei and locus coeruleus; the ventral and central areas of the RPO and locus coeruleus complex were especially rich in DOR-labeled somata. Within the vRPO, DOR was localized mainly in the cytoplasm and on plasma membranes of medium to large dendrites (47.8% of DOR-labeled profiles), which received both symmetric and asymmetric synaptic contacts mainly from non-labeled (82% of total inputs) axon terminals. Less frequently, DOR was distributed presynaptically in axon terminals (19% of DOR-labeled profiles). Our results suggest that DOR activation in vRPO regulates REM sleep occurrence by modulating postsynaptic responses to both excitatory and inhibitory afferents. DOR activation in vRPO could have, however, an additional role in direct modulation of neurotransmitter release from axon terminals.

Cranial visceral afferents activate central pathways that mediate systemic homeostatic processes. Afferent information arrives in the brainstem nucleus of the solitary tract (NTS) and is relayed to other CNS sites for integration into autonomic responses and complex behaviors. Little is known about the organization or nature of processing within NTS. We injected fluorescent retrograde tracers into two nuclei to identify neurons that project to sites involved in autonomic regulation: the caudal ventrolateral medulla (CVLM) or paraventricular nucleus of the hypothalamus (PVN). We found distinct differences in synaptic connections and performance in the afferent path through NTS to these neurons. Anatomical studies using confocal and electron microscopy found prominent, primary afferent synapses directly on somata and dendrites of CVLM-projecting NTS neurons identifying them as second-order neurons. In brainstem slices, afferent activation evoked large, constant latency EPSCs in CVLM-projecting NTS neurons that were consistent with the precise timing and rare failures of monosynaptic contacts on second-order neurons. In contrast, most PVN-projecting NTS neurons lacked direct afferent input and responded to afferent stimuli with highly variable, intermittently failing synaptic responses, indicating polysynaptic pathways to higher-order neurons. The afferent-evoked EPSCs in most PVN-projecting NTS neurons were smaller and unreliable but also often included multiple, convergent polysynaptic responses not observed in CVLM-projecting neurons. A few PVN-projecting NTS neurons had monosynaptic EPSC characteristics. Together, we found that cranial visceral afferent pathways are structured distinctly within NTS depending on the projection target. Such, intra-NTS pathway architecture will substantially impact performance of autonomic or neuroendocrine reflex arcs.

GABA mediates inhibitory effects in neurons of the ventral part of the oral pontine reticular nucleus (vRPO). Evidence increasingly suggests that GABA plays an important role in the modulation of rapid eye movement (REM) sleep generation in the cat vRPO. Here, we investigate the anatomical substrate of this modulation using GABA immunocytochemistry. Immunoperoxidase labeling revealed a few small GABA-immunoreactive cell bodies scattered throughout the vRPO. The numerical densities of all vRPO synapses and the GABA-immunoreactive synapses were estimated, at the electron microscopical level, by using a combination of the physical disector and the post-embedding immunogold techniques. We estimated that 30% of all vRPO synaptic terminals were immunoreactive to GABA. Our findings support the hypothesis that vRPO neuron activity is significantly controlled by inhibitory GABAergic terminals that directly target somata and the different parts of the dendritic tree, including distal regions. GABAergic input could inhibit vRPO REM sleep-inducing neurons during other states of the sleep-wakefulness cycle such as wakefulness or non-REM sleep.

We present a case of juvenile dermatomyositis with unusual histopathologic findings. The child presented with a course consistent with dermatomyositis, a diagnosis confirmed by finding reticulotubular aggregates in endothelial cells on electron microscopy. However, histopathology of his muscle biopsy revealed a striking pattern of glycogen accumulation, to an extent similar to that seen in glycogen storage diseases; this degree of accumulation could potentially confound histopathologic diagnosis.

The aim of this study was to investigate the ultrastructure of the reticular thalamic nucleus (RTN) in rats of WAG/Rij strain, an established model for human absence epilepsy. Most RTN neurons are medium-to large-sized and have either dark or light appearance, depending on their functional state. Moreover, small-sized neurons with short axons are present, their characteristics being described for the first time.

This study investigated the synaptic interactions between hypoglossal motoneurons that project to the genioglossus muscle and substance P (SP) containing immunoreactive nerve terminals. Cholera toxin B conjugated to horseradish peroxidase (CTB-HRP) was injected into the right half of the genioglossus muscle in four anesthetized cats. Two days later, the animals were perfused with acrolein fixative. Tetramethylbenzidine (TMB) was the chromogen used to detect retrogradely labeled cells containing CTB-HRP. The tissues were then processed for immunocytochemistry using an antiserum raised against SP with diaminobenzidine (DAB) as the chromogen. At the light microscopic level, labeled cells were observed primarily ipsilaterally in ventral and ventrolateral subdivisions of the hypoglossal nucleus. The majority of these labeled cells were observed at the level of the area postrema. At the electron microscopic level, SP-like immunoreactive nerve terminals formed synaptic contacts with retrogradely labeled dendrites and perikarya. Nineteen percent of the terminals that contacted retrogradely labeled cells contained SP. These are the first ultrastructural studies demonstrating synaptic interactions between protruder hypoglossal motoneurons and SP terminals. These studies demonstrate that hypoglossal motoneurons which innervate the major protruder muscle of the tongue, the genioglossus muscle, may be modulated by SP. Thus, SP may play a role in the control of protrusive movements of the tongue acting via neurokinin receptors.

The dorsal parvocellular reticular formation (PCRt) receives projection of the trigeminal mesencephalic nucleus neurons. It contains the dorsal group of interneurons that integrate and coordinate activity of the oral motor nuclei. Ultrastructural features of synaptic connection from the dorsal PCRt neurons to the motoneurons of the hypoglossal nucleus (XII) were examined at both the light and electron microscopic levels in rats. Biotinylated dextran amine (BDA) was initially iontophoresed into the dorsal part of PCRt unilaterally. Seven days later horseradish peroxidase (HRP) was injected into the body of the tongue. After histochemical reaction for visualization of HRP and BDA, the BDA-labeled fibers and terminals were seen distributing bilaterally in XII with ipsilateral predominance. BDA-labeled terminals were closely apposed upon HRP retrogradely labeled somata and dendrites of the XII motoneurons. A total of 1408 BDA-labeled boutons were examined ultrastructurally, which had mean size of 1.22+/-0.37 microm in diameter. Five hundred-ninety three of these boutons in both the ipsilateral (n=401) and contralateral (n=192) XII were seen to synapse on both the dendrites and somata of HRP-labeled motoneurons. The vast majorities of synapses were axodendritic (98%, 580/593), while 2% of them were axosomatic. Of the 1408 BDA-labeled boutons, 69.6% of them were S-type boutons containing small clear and spherical synaptic vesicles and 30.4% of them were PF-type boutons containing pleomorphic and flattened synaptic vesicles. Approximately 64% of synapses between BDA-labeled boutons and HRP-labeled motoneurons were asymmetric, and 33% of synapses were symmetric. No axoaxodendritic or axoaxosomatic synaptic triad was observed. The present study illustrated the anatomical pathway and synaptological characteristics of neuronal connection between the dorsal PCRt premotor neurons and the XII motoneurons. Its functional significance in coordinating activity of XII motoneurons during oral motor behaviors has been discussed.

The giant reticulospinal synapse in lamprey provides a unique model to study synaptic vesicle traffic. The axon permits microinjections, and the active zones are often separated from each other, which makes it possible to track vesicle cycling at individual release sites. However, the proportion of reticulospinal synapses with individual active zones ("simple synapses") is unknown and a quantitative description of their organization is lacking. Here, we report such data obtained by serial section analysis, intermediate-voltage electron microscopy, and electron tomography. The simple synapse was the most common type (78%). It consisted of one active zone contacting one dendritic process. The remaining synapses were "complex," mostly containing one vesicle cluster and two to three active zones synapsing with distinct dendritic shafts. Occasional axosomatic synapses with multiple active zones forming synapses with the same cell were also observed. The vast majority of active zones in all synapse types contained both chemical and electrotonic synaptic specializations. Quantitative analysis of simple synapses showed that the majority had active zones with a diameter of 0.8-1.8 microm. The number of synaptic vesicles and the height of the vesicle cluster in middle sections of serially cut synapses correlated with the active zone length within, but not above, this size range. Electron tomography of simple synapses revealed small filaments between the clustered synaptic vesicles. A single vesicle could be in contact with up to 12 filaments. Another type of filament, also associated with synaptic vesicles, emerged from dense projections. Up to six filaments could be traced from one dense projection., (Copyright 2002 Wiley-Liss, Inc.)

Ligands of the delta-opioid receptor tonically influence sympathetic outflow. Some of the actions of delta-opioid receptor agonists may be mediated through C1 adrenergic neurons in the rostral ventrolateral medulla. The goal of this study was to determine whether C1 adrenergic neurons or their afferents contain delta-opioid receptors. Single sections through the rostral ventrolateral medulla were labeled for delta-opioid receptor using the immunoperoxidase method and the epinephrine synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) using the immunogold method, and examined at the light and electron microscopic level. Few ( approximately 5% of 903) profiles dually labeled for PNMT and delta-opioid receptor were detected; most of these were dendrites with diameters < 1.5 microm. delta-Opioid receptor immunoreactivity was affiliated with multivesicular bodies in dually labeled perikarya, whereas delta-opioid receptor immunoperoxidase labeling appeared as isolated clusters within both singly and dually labeled dendrites. The majority ( approximately 83% of 338) of delta-opioid receptor-immunoreactive profiles were axons and axon terminals. delta-Opioid receptor-immunoreactive terminals averaged 0.75 microm in diameter, contained numerous large dense-core vesicles and usually formed appositions or asymmetric (excitatory-type) synapses with their targets. The majority (>50% of 250) of delta-opioid receptor-immunoreactive axons and axon terminals contacted PNMT-immunoreactive profiles. Most of the contacts formed by delta-opioid receptor-immunoreactive profiles ( approximately 75% of 132) were on single-labeled PNMT-immunoreactive dendrites with diameters <1.5 microm. The prominent localization of delta-opioid receptors to dense-core vesicle-rich presynaptic profiles suggests that delta-opioid receptor activation by endogenous or exogenous agonists may modulate neuropeptide release. Furthermore, the presence of delta-opioid receptors on axon terminals that form excitatory-type synapses with PNMT-immunoreactive dendrites suggests that delta-opioid receptor ligands may modulate afferent activity to C1 adrenergic neurons. The observation that some PNMT-immunoreactive neurons contain delta-opioid receptor immunoreactivity associated with multivesicular bodies and other intracellular organelles suggests that some C1 adrenergic neurons may present, endocytose and/or recycle delta-opioid receptors.

Axons containing serotonin descend from brainstem to spinal cord and are thought to contribute to stimulation-produced and opioid analgesia, partly by a direct inhibitory action of serotonin on projection neurones. The density of serotoninergic innervation is highest in lamina I, which contains many nociceptive projection neurones. Two sets of anatomical criteria have been used to classify lamina I projection neurones: somatodendritic morphology and presence or absence of the neurokinin 1 receptor. To test whether the strength of serotoninergic innervation of lamina I projection neurones was related to morphology or neurokinin 1 receptor expression, we used confocal microscopy to determine the density of serotoninergic contacts on 60 cells retrogradely labelled from the caudal ventrolateral medulla. The contact density on neurones with the neurokinin 1 receptor was variable, with some cells receiving heavy input and others having few contacts. However, on average they received significantly more contacts (5.64 per 1000 microm(2) plasma membrane +/- 0.47, S.E.M.) than neurones which lacked the receptor (2.49 +/- .36). Among the neurokinin 1 neurones, serotoninergic innervation density was not related to morphology. Since the majority of serotoninergic boutons in lamina I of rat spinal cord do not appear to form synapses, we carried out electron microscopy on three heavily innervated neurokinin 1 receptor-immunoreactive projection neurones. Symmetrical synapses were found at 89% of serotoninergic contacts. These results indicate that serotoninergic innervation of lamina I projection neurones in the rat spinal cord is related to expression of neurokinin 1 receptors, but not to morphology, and that (at least on heavily innervated neurones) most serotonin-containing boutons which are in contact form synapses.

Neuronal composition was studied in two human thalamic nuclei--nucleus ventralis anterior and nucleus ventralis lateralis using serial horizontal and frontal sections stained using Kluver-Barrera and Golgi silver impregnation methods. It was found that the number of neuronal types, composing the nuclei (equal to eight) is greater than previously reported. Proposed neuronal classification based on the characteristics of their processes, in comparison with a similar study performed in dogs (pups) permitted to distinguish two types of neurons--long-axon and short-axon. Long-axon neurons are subdivided into sparsely-branched (reticular and short-dendritic) and densely-branched (arborescent, giant and medium bush-like, penicillar varieties). Short-axon neuronal type includes smooth-dendritic and "shaggy" cells.

Central nervous system oxygen toxicity (CNS O2 toxicity) is preceded by release of hyperoxic vasoconstriction, which increases regional cerebral blood flow (rCBF). These increases in rCBF precede the onset of O2-induced convulsions. We have tested the hypothesis that hyperbaric oxygen (HBO2) stimulates NO* production in the brain that leads to hyperemia and anticipates electrical signs of neurotoxicity. We measured rCBF and EEG responses in rats exposed at 4 to 6 atmospheres (ATA) of HBO2 and correlated them with brain interstitial NO* metabolites (NO(x)) as an index of NO* production. During exposures to hyperbaric oxygen rCBF decreased at 4 ATA, decreased for the initial 30 min at 5 ATA then gradually increased, and increased within 30 min at 6 ATA. Changes in rCBF correlated positively with NO(x) production; increases in rCBF during HBO2 exposure were associated with large increases in NO(x) at 5 and 6 ATA and always preceded EEG discharges as a sign of CNS O2 toxicity. In rats pretreated with L-NAME, rCBF remained maximally decreased throughout 75 min of HBO2 at 4, 5 and 6 ATA. These data provide the first direct evidence that increased NO* production during prolonged HBO2 exposure is responsible for escape from hyperoxic vasoconstriction. The finding suggests that NO* overproduction initiates CNS O2 toxicity by increasing rCBF, which allows excessive O2 to be delivered to the brain.

Physiological studies have suggested that mu-opioid receptor (MOR) activation can both excite and inhibit reticulospinal neurons in the rostral ventrolateral medulla (RVL), possibly via influences on GABAergic neurons. Thus, to determine the cellular relationships of MORs to GABAergic neurons in the RVL, two experimental approaches were used. First, single sections through the RVL were labeled for MOR using immunoperoxidase detection and for GABA using immunogold detection and examined by electron microscopy. These studies revealed that MOR-immunoreactive (IR) terminals were smaller on average than GABA-IR terminals and formed both asymmetric and symmetric synapses, whereas GABA-IR terminals formed exclusively symmetric synapses. MOR and GABA immunoreactivities rarely co-localized. Interactions between axons and terminals containing MOR or GABA immunoreactivity were primarily: (1) direct appositions with each other; or (2) convergence onto a common dendritic target that sometimes contained either MOR or GABA immunoreactivity. Since the identity of these target dendrites mostly was unknown, a second study was designed to determine if they might be reticulospinal neurons. For this study, reticulospinal neurons were identified with a retrograde tracer and both MOR and GABA were localized in the same sections of the RVL. These studies revealed that numerous GABA-IR terminals formed symmetric synapses on the perikarya and proximal dendrites of reticulospinal neurons. In contrast, few MOR-IR terminals contacted reticulospinal perikarya and large dendrites although they were often found nearby. These results provide anatomical evidence that MOR activation by endogenous or exogenous agonists may indirectly alter GABAergic neurotransmission in the RVL either through presynaptic interactions between cells or through competing influences on postsynaptic targets.

Quantitative characteristics (the area and number of chromocenters) of the interphase C-heterochromatin in the nuclei of pyramidal neurons of the midbrain reticular formation, sensorimotor cortex, and hippocampus (CA3) of rat strains with different genetically determined excitability were studied in the normal state of the animals and after exposure to a short-term emotional pain stress. The results indicate a relationship between the excitability of the nervous system and structural-functional state of the neuronal interphase heterochromatin. The role of cytogenetic features of different brain structures in the CNS functioning and behavior and their relation with genetically determined excitability of the nervous system are discussed.

The formation of synapses in ontogenesis is an important problem of neuromorphology. The paper shows that the bulk of synapses of the developing brain is formed on the basis of previous specialization of membranes. Early in ontogenesis, most formed synapses shows asymmetric contacts. In postnatal ontogenesis, the formation of synapses proceeds due to the simultaneous occurrence of specialization of membranes and synaptic vesicles, by forming a "dot" active zone. Systemogenesis, such as the general law of brain development, plays an important role in understanding the functional significance of the shown mechanisms of maturation of synapses.

The activity of bulbospinal (presympathetic) vasomotor neurons of the rostral ventrolateral medulla is modulated pre- and postsynaptically by exogenously applied opioid agonists. To determine whether these neurons receive direct opioid inputs, we examined the relationship between bulbospinal barosensitive neurons and nerve terminals immunoreactive for enkephalin in the rostral ventrolateral medulla of rats. By light microscopy, we mapped the distribution of close appositions by enkephalin-immunoreactive varicosities on 10 bulbospinal barosensitive neurons labelled in vivo with biotinamide. We also examined four labelled neurons ultrastructurally for synapses by enkephalin-immunoreactive terminals and determined with post-embedding immunogold labelling whether these enkephalin-positive terminals contained amino acids. Enkephalin-immunoreactive varicosities closely apposed all bulbospinal barosensitive neurons. Maps of the dendritic distribution of appositions indicated that fast-conducting bulbospinal barosensitive neurons with myelinated axons (conduction velocity >3 m/s; n=3) received many appositions (up to 470/neuron); and slowly conducting neurons with unmyelinated axons (conduction velocity <0.90 m/s; n=3), substantially fewer. Ultrastructural analysis of three fast- and one slowly conducting bulbospinal barosensitive neurons revealed numerous synapses from enkephalin-immunoreactive terminals on cell bodies and dendrites. Enkephalin-positive terminals synapsing on bulbospinal barosensitive neurons contained one or more amino acid: GABA+glycine, glutamate alone or GABA+glutamate. Enkephalin-immunoreactive terminals located near biotinamide-labelled cells contained a similar variety of amino acids. In summary, enkephalin-immunoreactive terminals in the rostral ventrolateral medulla densely innervate lightly myelinated presympathetic neurons and more sparsely those with unmyelinated axons. Enkephalin is present in both excitatory (glutamate-immunoreactive) and inhibitory (GABA- and/or glycine-immunoreactive) terminals. The data suggest that endogenous enkephalin inhibits amino acid release from terminals that innervate bulbospinal barosensitive neurons of the rostral ventrolateral medulla.

In an attempt to contribute to the current knowledge of the brainstem reticular formation synaptic organization, the ultrastructure and distribution of synaptic terminal profiles on neurons in the ventral part of the oral pontine reticular nucleus (vRPO), the rapid eye movement (REM) sleep-induction site, were studied quantitatively. Terminals with asymmetric contacts and rounded vesicles were classified according to vesicle density as type I or II (high or low density, respectively). The area, apposed perimeter length, and mitochondrial area of type I terminals, on average, were significantly smaller than those of type II terminals. Type III and IV terminals had symmetric contacts and oval and/or flattened vesicles; type III terminals formed synapses between them and on initial axons. Type V and VI terminals showed characteristics intermediate to those of asymmetric and symmetric synapses. Interestingly, some terminal features were related to both terminal area and postsynaptic dendritic diameter. The percentages of different synapses sampled on somata were as follows: asymmetric synapses (usually formed by type II terminals; mean +/- S.D.), 26.4% +/- 3%; symmetric synapses, 46.7% +/- 5.2%; and intermediate synapses, 26.9% +/- 6.1%. The percentages of different synapses sampled on dendrites were asymmetric synapses, 62.1% +/- 9%; symmetric synapses, 25.6% +/- 8.1%; and intermediate synapses, 12.3% +/- 1.7%. Comparison between large- and small-diameter dendrites revealed that the percentages of symmetric synapses and type II terminals decreased, whereas the percentages of type I terminals increased as postsynaptic dendritic diameters became smaller. Synaptic density was approximately four times lower on somata than on dendrites. The vRPO synaptic organization reflects some patterns that are similar to those found in other regions of the central nervous system as well as specific synaptic patterns that are probably related to its functions: the generation and maintenance of REM sleep and the control of eye movement or limb muscle tone., (Copyright 2000 Wiley-Liss, Inc.)

Calbindin-D28k (calbindin) is a calcium-binding protein that is distributed widely in the rat brain. The localisation of calbindin immunoreactivity in the medulla oblongata and its colocalisation with adrenaline-synthesising neurons [phenylethanolamine-N-methyltransferase-immunoreactive (PNMT-IR)] was examined (Granata and Chang [1994] Brain Res. 645:265-277). However, detailed information about the distribution of calbindin-IR neurons in the reticular formation of the medulla oblongata in particular is lacking. In this report, the authors address this issue with an emphasis on the quantitation of calbindin-IR neurons, catecholamine neurons [tyrosine hydroxylase (TH)-IR, or PNMT-IR], and spinally projecting neurons in the ventral brainstem. Rats received injections of the retrograde tracing agent cholera toxin B (CTB) into the thoracic spinal cord or into the superior cervical ganglion. Immunocytochemistry was used to reveal calbindin, TH, PNMT, and CTB immunoreactivity. Ten calbindin-IR cell groups were identified within the pontomedullary reticular formation. Seven previously undescribed but distinct clusters of calbindin-IR neurons were found. Within the ventral pons, a population of calbindin-IR neurons occurred dorsal but adjacent to the A5 cell group. These calbindin-IR neurons did not contain either TH or PNMT immunoreactivity, and few if any of these neurons projected to the spinal cord. A distinct group of calbindin-IR neurons was present in the ventral medulla. Seventy-five percent of these calbindin-IR neurons contained TH immunoreactivity, 45% contained PNMT immunoreactivity, and 21% were spinally projecting neurons. Spinally projecting, calbindin-IR neurons were a subpopulation of PNMT-IR cells. In the caudal ventral medulla, no TH-IR or PNMT-IR cells were calbindin-IR. In the intermediolateral cell column, close appositions of calbindin-IR terminals on identified sympathetic preganglionic neurons as well as calbindin-IR synapses indicated that these neurons may affect directly the sympathetic outflow. The results demonstrate for the first time the existence of a new subpopulation of spinally projecting, PNMT-IR neurons in the rostral ventrolateral medulla., (Copyright 2000 Wiley-Liss, Inc.)

Before action potential-evoked Ca2+ transients, basal presynaptic Ca2+ concentration may profoundly affect the amplitude of subsequent neurotransmitter release. Reticulospinal axons of the lamprey spinal cord receive glutamatergic synaptic input. We have investigated the effect of this input on presynaptic Ca2+ concentrations and evoked release of neurotransmitter. Paired recordings were made between reticulospinal axons and the neurons that make axo-axonic synapses onto those axons. Both excitatory and inhibitory paired-cell responses were recorded in the axons. Excitatory synaptic inputs were blocked by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) and by the NMDA receptor antagonist 2-amino-5-phosphonopentanoate (AP-5; 50 microM). Application of NMDA evoked an increase in presynaptic Ca2+ in reticulospinal axons. Extracellular stimulation evoked Ca2+ transients in axons when applied either directly over the axon or lateral to the axons. Transients evoked by the two types of stimulation differed in magnitude and sensitivity to AP-5. Simultaneous microelectrode recordings from the axons during Ca2+ imaging revealed that stimulation of synaptic inputs directed to the axons evoked Ca2+ entry. By the use of paired-cell recordings between reticulospinal axons and their postsynaptic targets, NMDA receptor activation was shown to enhance evoked release of transmitter from the axons that received axoaxonic inputs. When the synaptic input to the axon was stimulated before eliciting an action potential in the axon, transmitter release from the axon was enhanced. We conclude that NMDA receptor-mediated input to reticulospinal axons increases basal Ca2+ within the axons and that this Ca2+ is sufficient to enhance release from the axons.

This study investigated whether the topographic differences in the functional properties of the tectal motor map of goldfish are related to particular patterns of connections with downstream structures. With this aim, the distribution of synaptic boutons in the mesencephalic and rhombencephalic structures was studied after discrete injections of the tracer biotinylated dextran amine were placed at separate sites along the tectal anteroposterior axis. Irrespective of the location of the injection site, the boutons were more abundant in the mesencephalon than in the rhombencephalon, and they were located chiefly ipsilaterally all throughout the brainstem. In the mesencephalon, the boutons were found in its ventrolateral reticular formation and, to a lesser extent, in the nucleus of the medial longitudinal fasciculus, the oculomotor and isthmi nuclei, and the torus semicircularis. In the mesencephalic reticular formation, the bouton location was distributed topographically with respect to the injection site. Terminals were also observed in the nucleus of the medial longitudinal fasciculus after injections into anteromedial or middle tectal zones. In the oculomotor nucleus, boutons were present exclusively in the case of the anteromedial injection. In the rhombencephalon, most boutons were found in the superior reticular formation, and their number decreased in the medial and inferior reticular formations. A topographic distribution could be observed within the superior reticular formation, although its density was attenuated compared with that observed in the mesencephalic reticular formation. The domains of synaptic endings on the ipsilateral side were different from those on the contralateral side: The ipsilateral synaptic endings were located more medially. Finally, a few boutons were also found in the vestibulocerebellar area on either the ipsilateral or the contralateral side, depending on the injection site. From these data, the authors conclude that, in goldfish, irrespective of the tectal injection site, the endings are in similar nuclei in the brainstem; however, the distribution of synaptic boutons within such nuclei can be related to the functional properties of each tectal zone.

We examined a nigro-tecto-bulbar pathway to the parvicellular reticular formation (RFp), where many premotor neurons for orofacial motor nuclei are known to be distributed, by using a combined anterograde and retrograde tracing method. After contralateral injections of biotinylated dextranamine (BDA) into the dorsolateral part of the substantia nigra (SNr) and cholera toxin B subunit (CTb) into the RFp, overlapping distributions of BDA-labeled terminals and CTb-labeled neuronal cell bodies were found in the lateralmost part of the superior colliculus (SC) ipsilateral or contralateral to the site of BDA injection or CTb injection, respectively. After contralateral injections of BDA into the SNr and horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) injection into the RFp, ipsilateral labeled axon terminals with BDA were found to make symmetrical synaptic contacts with the somata and dendrites of contralateral labeled neurons with WGA-HRP in the lateralmost part of the SC. Furthermore, we demonstrated that BDA-labeled axon terminals were immunoreactive for GABA, by using the anterograde tracing method combined with immunohistochemistry for GABA. Thus, GABA-like immunoreactive fibers originating from the dorsolateral part of the SNr make monosynaptic contacts with the tectal neurons sending their axons to the RFp.

The perireticular nucleus lies among the fibres of the internal capsule and in the rat is relatively large at birth. Like the subplate of the neocortex, it is dramatically reduced in the adult (Mitrofanis, 1992). On embryonic day 17 (E17), cells of the perireticular nucleus project to the cortex (Adams and Baker, 1995); at birth there is also a projection to the thalamus (Mitrofanis and Baker, 1993). When corticofugal axons reach the perireticular nucleus, they separate into the descending tracts and the corticothalamic pathway. The axons then enter the internal capsule on their way to the thalamus, defasciculate and contribute to a complex zone of interweaving fibres seen in this region (Adams and Guillery, 1994). We have found that the cells of the perireticular nucleus are immunopositive for an antibody (3A10) to a phosphorylated neurofilament protein (Yamada et al., 1991), and we have shown that these 3A10-positive perireticular cells extend their dendrites across the path of cortical fibres at the earliest age studied (E17). We have confirmed this dendritic orientation by Golgi staining at a later stage (P3). Electron micrographs show that axo-somatic and axo-dendritic synapses first appear in the perireticular nucleus at about the day of birth. Prenatally, between E14 and E19, the major specialized membrane interrelationships seen are omega formations between adjacent profiles that often contain vesicles. Omega formations are rarely seen at later stages. Puncta adhaerentia typified by opposing membrane densities with no associated vesicles were seen at all ages studied. We suggest that early membrane contacts (omega formations) may represent transient organisational or guidance influences since they occur during a period when axonal pathways are being defined.

The postnatal development of inhibitory GABAergic circuits in the thalamic reticular and ventrobasal nuclei was studied in rats ranging from the day of birth to the end of the third postnatal week by means of a postembedding immunogold staining procedure to visualize GABA. In the reticular nucleus, GABA labeling was present from birth in cell bodies, dendrites, growth cones and a few synaptic terminals, whereas in the ventrobasal nucleus it was exclusively in axonal processes identifiable as growth cones, vesicle-rich profiles and synaptic terminals. In both nuclei, GABA-labeled synaptic terminals were, however, very scarce and immature in neonatal animals and they became numerous and morphologically mature only after the end of the second postnatal week. These findings suggest that inhibitory synaptic responses in the somatosensory thalamus are not yet fully mature throughout the first two postnatal weeks and support the hypothesis that GABA may initially play trophic roles. The relatively late maturation of the thalamic GABAergic system may have important functional consequences, as the reticulothalamic circuits are responsible for the generation of spindle wave oscillations whose cellular mechanisms are also involved in the generation of spike-and-wave (absence) seizures in humans and in animal models.

In the present study, we asked whether supraspinal axons grow through a complete transection of the spinal cord in the developing opossum Didelphis virginiana. When the thoracic cord was transected at postnatal day (PD) 5 and bilateral injections of Fast Blue (FB) were made four segments caudal to the lesion 30-40 days later, FB-containing neurons were found in each of the supraspinal nuclei labeled by comparable injections in age-matched unlesioned controls. Continuity between the cut ends of the cord was obviously gross when the animals were killed, and histologically recognizable spinal cord was present at the lesion site. When the same procedure was followed on pups subjected to transection at PD12, FB-containing neurons were still present at supraspinal levels, but they appeared to be fewer in number than in the PD5 cases or the age-matched controls, and none were found within the medial pontine reticular and lateral vestibular nuclei. When the lesion was made at PD20, labeled neurons were even fewer in number, and when it was made at PD26, they were restricted to the medullary raphe and the red nuclei. There was no evidence for growth of supraspinal axons across lesions made at PD33. We conclude that supraspinal axons grow through the lesion after transection of the spinal cord in neonatal opossums and that the critical period for growth of reticulospinal and vestibulospinal axons through the lesion ends earlier than that for comparable growth of raphespinal and rubrospinal axons.

Electron microscopy has been employed to analyze the normal maturational sequence that characterizes the postnatal development of synaptic circuits in the ventrobasal (VB) and reticular (Rt) thalamic nuclei of rats at different ages (from birth to the end of the third postnatal week). Throughout the first postnatal week, similar signs of immaturity are observed in both nuclei, mainly consisting in scarcity of cytoplasmic organelles, presence of wide extracellular spaces, and absence of myelinated fibers. Several synaptic terminals are however present from birth, thus indicating that some of the afferents have already reached and contacted their thalamic target during embryonic life. Most of the terminals are small and contain only a few round, clear vesicles, and therefore their cytological features do not allow the identification of their origin. In particular, in both nuclei, terminals with flat vesicles and symmetric specialization are only rarely observed, and in VB the ascending terminals are not distinguishable from terminals of other sources as they are in adults. During the second postnatal week, progressive maturational changes in VB and Rt lead to neurons having well-developed cytoplasmic organelles and to an elaborate neuropil containing myelinated fibers and synaptic terminals that are morphologically heterogeneous and resemble the adult ones. The permanence of growth cone-like profiles and of numerous somatic and dendritic protrusions, often contacted by synaptic terminals, indicates that a certain degree of reorganization is still taking place in both nuclei. By the end of the third postnatal week the synaptic organization of VB and Rt is indistinguishable from that observed in adults. This ultrastructural study shows that the appearance of the neuropil of VB and Rt and the morphological complexity of the synaptic arrangements characteristic of the adult rat are not present in neonates, but are gradually acquired during the first three postnatal weeks, and that they result from progressive modifications in circuit organization involving both pre- and postsynaptic elements.

Neurofilaments of the sea lamprey are unique in being homopolymers of a single subunit (NF-180). Digoxigenin-labeled RNA probes complementary to NF-180 were used to determine the distribution and timing of expression of neurofilament message in the brain and spinal cord of the lamprey. In the brainstem, detection of NF-180 mRNA was restricted to neurons with axons projecting to the spinal cord or the periphery. The majority of brainstem neurons, whose axons project locally, did not express NF-180 within the detection limits of this technique. NF-180-positive neurons included cells with a wide range of axon diameters, suggesting neurofilament mRNA expression was linked to axon length rather than caliber. To further evaluate this hypothesis, expression was studied in animals of different developmental stages between larvae and adults. In younger (shorter) larvae, the large Mauthner and rhombencephalic Müller cells did not express NF-180 mRNA, even though their axons are among the largest caliber in the animal and extend the entire length of the spinal cord. In contrast, many other reticulospinal neurons, whose axons are smaller in diameter than those of the Müller and Mauthner cells, expressed NF-180 message throughout larval development. Furthermore, neurons of the cranial motor nuclei did not express NF-180 until later developmental stages and the extraocular motor neurons did not label until metamorphosis. Therefore, while detectable neurofilament mRNA expression in the lamprey is restricted to neurons with long axons, its expression in this population of neurons appears to be developmentally regulated by factors still not determined. It is postulated that need for NF message is determined by a balance between the volume of axon to be filled and the rate of turnover of NF in that axon.

Single axons of tectospinal (TS) and reticulospinal (RS) neurons were stained with intraaxonal injection of HRP after electrophysiological identification, and their axonal trajectory was reconstructed at C1-C3 of the cat. TS neurons were located in the intermediate or deep layers of the caudal two-thirds of the superior colliculus (SC) and had multiple axon collaterals (up to seven collaterals) per stem axon). Collaterals had a simple structure, ramified several times mainly in the transverse plane, and terminated in the lateral parts of laminae V-VIII. More than half also had terminals in lamina IX. Terminals of TS neurons did not appear to make contacts with either the somas or proximal dendrites of retrogradely-labeled motoneurons in lamina IX, but clear contacts were found on counterstained interneurons in the lateral part of laminae V-VIII. Here, we examined three stained spinal interneurons receiving monosynaptic excitation from the SC. These interneurons had multiple axon collaterals mainly in laminae VII-IX, and made extensive contacts with retrogradely-labeled motoneurons of multiple neck muscles. Stem axons of single RS neurons receiving input from the contralateral SC ran in the ventromedial funiculus and gave off multiple axon collaterals to laminae VII-IX over at least several cervical segments. Their terminal boutons appeared to make contact with both the somas and proximal dendrites of retrogradely-labeled neck motoneurons. Single RS neurons made contacts with motoneurons of different neck muscles. These results provide evidence for functional synergies at the level of single RS neurons and spinal interneurons for neck movements. The present finding indicates that the direct TS projection to the spinal cord may influence the activity of multiple neck muscles mainly via spinal interneurons, and plays an important role in control of head movement in parallel with the tecto-reticulospinal system.

Microinjection of L-glutamate into a restricted area of the medullary gigantocellular reticular formation, the gigantocellular depressor area (GiDA), lowers arterial pressure. Unlike the nuclei tractus solitarii and the caudal ventrolateral medulla, the two principle medullary vasodepressor areas, the GiDA projects directly to the spinal cord and not to the rostral ventrolateral medulla (Aicher et al. [1994] Neuroscience 60:761-779). We investigated whether neurons within GiDA directly innervate autonomic areas of the thoracic spinal cord. Fluoro-Gold injected into the thoracic spinal cord labeled neurons within functionally defined vasodepressor sites in the GiDA in the same animal. To examine the morphology of GiDA efferents to the spinal cord, the anterograde tracer Phaseolus vulgaris-leucoagglutinin was iontophoresed into the GiDA, and efferent processes in the intermediolateral cell column and nucleus intercalatus spinalis were examined by electron microscopy. Labeling was confined to axons and axon terminals (n = 144) that usually contained primarily small clear vesicles, contacted large and small dendrites, and formed symmetric (inhibitory) synapses. To determine whether some of the postsynaptic targets of GiDA efferent terminals in the thoracic spinal cord were sympathoadrenal preganglionic neurons, these neurons were retrogradely labeled from the adrenal gland with Fluoro-Gold in rats that had deposits of the anterograde tracer, biotinylated dextran amine (BDA), in the GiDA. Some BDA-containing terminals formed symmetric synapses with dendrites containing Fluoro-Gold. We conclude that a population of neurons in the GiDA monosynaptically innervates some sympathetic preganglionic neurons. The findings suggest the presence of a novel reticulospinal sympathoinhibitory projection originating in the GiDA.

Intracellular staining with biotinamide was used to study the axonal projection and synaptic morphology of rat jaw-muscle spindle afferents. Intracellular recordings in the mesencephalic trigeminal nucleus (Vme) were identified as spindle afferent responses by their increased firing during stretching of the jaw-elevator muscles. Biotinamide-stained axon collaterals with boutons were found in the trigeminal motor nucleus (Vmo), Vme, the region dorsal to Vmo including the supratrigeminal region, the dorsomedial portion of the trigeminal principal sensory nucleus, and the dorsomedial part of the rostral spinal trigeminal subnucleus oralis. Additional, previously undescribed projections of jaw-muscle spindle afferents were found to the dorsomedial portion of the caudal spinal trigeminal subnucleus oralis (Vodm), the dorsomedial part of the spinal trigeminal subnucleus interpolaris (Vidm), the caudal parvicellular reticular formation, laminae IV and V of the spinal trigeminal subnucleus caudalis (Vc), and the dorsal division of the medullary reticular field. Labeled spindle boutons in Vodm formed predominately axodendritic synapses. Some of these boutons received presynaptic inputs from unlabeled P-type boutons containing clear, spherical, or flattened vesicles. In Vidm, labeled collaterals and boutons were densely clustered into glomerular-like structures. Labeled boutons in Vidm made axodendritic, axosomatic, and axoaxonic synapses and received synaptic contacts from unlabeled boutons containing clear, spherical, or flat and pleomorphic vesicles. Unlabeled presynaptic boutons in Vidm occasionally contained dense core vesicles. Labeled boutons in Vc mainly formed synaptic contacts with large diameter dendrites. This projection of jaw-muscle spindle afferents to caudal brainstem regions may play a significant role in masticatory-muscle stretch reflexes and in the integration of trigeminal proprioceptive information and its transmission to higher centers.

This study was aimed at determining the synaptic circuitry that contributes to the alterations in thalamic function that accompany changes in behavioral states. The somatosensory sector of the thalamic reticular nucleus (RTN) was identified by microelectrode recording in cats and injected with Phaseolus vulgaris-leucoagglutinin (PHA-L). The axons of labeled RTN cells gave rise to collaterals within the RTN and continued into the dorsal thalamus where they terminated predominately in the ventral posterior lateral nucleus (VPL). After small injections in the upper limb representation of RTN, most labeled terminations in VPL were confined to its medial part, suggesting the presence of a topographic organization in the projection. Terminations were concentrated in localized, focal aggregations of boutons. Combined electron microscopic immunocytochemistry, using immunogold labeling for gamma-aminobutyric acid (GABA), showed that the PHA-L labeled boutons were GABA-positive terminals that ended in symmetrical synapses. Eighty-two percent of these synapses were on dendrites of relay neurons, 8.5% on dendrites of interneurons, and 9.3% on somata. The terminals of RTN axons form the majority of axon terminals ending in symmetrical synapses in VPL. Their concentration on relay neurons probably underlies the capacity of the RTN projection to reduce background activity of VPL relay neurons in the awake state and to maintain oscillatory behavior of these neurons in drowsiness and early phases of sleep.

Techniques for impregnation with ammoniacal silver carbonate provide valuable information on all types of tissue; however, the time investment required to impregnate a few sections has limited their application. We have shortened the impregnation times by using microwaves in techniques for reticular fibers, astrocytes, nerve fibers and chromaffin cells. The results were satisfactory with markedly reduced impregnation time and elimination of nonspecific silver deposits.

To locate a taste-related region in the thalamic reticular nucleus (Rt), we explored a reticular region having connections with the thalamic taste relay and the cortical taste area (CTA), by neuronal tracing methods using HRP, WGA-HRP and biocytin. Tracer injections at the thalamic taste relay, i.e., the parvicellular part of the thalamic posteromedial ventral nucleus (VPMpc), labeled cell bodies and axon terminals in a confined portion of the ipsilateral Rt. This was located at the ventromedial-most portion of the nucleus at the level of approx. 1.2 mm rostral to the rostral end of the VPMpc. Tracer injections at the CTA ipsilaterally labeled axon terminals in the same region of the Rt, as did the thalamic injections. When WGA-HRP was injected at this taste-related region in the Rt, we observed labeled cell bodies in layer VI in the CTA; in the VPMpc we saw densely labeled axon terminals but few, if any, labeled cell bodies. The results indicated that the taste-related region in the Rt receives input from the VPMpc and CTA, and sends output to the VPMpc. They also showed that the reticulo-thalamic projections were much heavier than the thalamo-reticular ones in the taste system.

The present study localised the superior salivatory nucleus (SSNc) in the reticular formation in rats using the retrograde axonal transport of fluorogold (FG). An ultrastructural study using the retrograde transport of horseradish peroxidase (HRP) was also carried out. After FG or cholera toxin conjugated-HRP (CT-HRP) injections into the lingual nerve, the labelling of SSNc in both the experiments was comparable. The retrogradely labelled cells were located in the ipsilateral parvocellular reticular formation (PCRt), dorsolateral to the facial nucleus and medial to the nucleus of the spinal tract of the trigeminal nerve. The rostrocaudal extent of the nucleus was about 0.9-1.05 mm, between the levels of the ascending facial nerve fibres caudally and the root of the facial nerve rostrally. The number of cells labelled was between 300-360, most of them being small to medium sized (14 x 22 mm) and appeared fusiform or polygonal. In rats receiving intraglandular injections of the tracers, the localisation and the rostrocaudal extent of the SSNc were similar, but the number of its containing labelled cells was only about 1/2 of the above. The SSNc cells that supplied the intra-glandular submandibular ganglion were not topographically arranged. On electron microscopy, the SSNc cells showed the typical features of preganglionic autonomic neurons. The cells contained a prominent round or oval nucleus. Their cytoplasm was rich in organelles especially conspicuous was the aggregations of the cisternae of rough endoplasmic reticulum in distinct clumps. In the neuropil between the SSNc cells were present numerous dendrites, axons, axon terminals and neuroglial cells. Most of the axon terminals contained predominantly round agranular vesicles with a few dense-cored or flattened agranular vesicles. The majority of the synapses associated with the HRP-labelled SSNc cells were on the dendrites with occasional axosomatic or axoaxonal synaptic contacts. A remarkable feature was the occurrence of synaptic contacts between HRP-labelled axons and some labelled soma.

The startle response is a bilateral response even when elicited by unilateral acoustic or tactile stimuli. Similarly, unilateral electrical stimulation of the reticular formation also elicits a bilateral startle-like response. To examine whether crossed reticular formation connections can distribute the effects of unilateral stimulation across the midline, we delivered one pulse to the caudal pontine (RPC) or medullary reticular formation (MRF) and a second pulse to the opposite side of the brain, at various interpulse intervals. The symmetric collision effects suggest that axons which produce at least 37% (range 23-53%) of the startle response efficacy cross from RPC to RPC with a mean conduction velocity of 13 m/s. Similar collision effects were observed between RPC and MRF sites but at shorter conduction times. To examine which axons might cause these collision effect, the axonally transported label DiI was injected post mortem into 37 RPC sites. Many coarse axons were observed to cross in fascicles between bilateral RPC sites and then separate in the contralateral RPC. The fiber diameters and trajectories of these DiI-labelled axons are consistent with the conduction velocities and trajectories of the substrates mediating the startle-like response determined in collision tests.

The distribution of synaptic terminals was quantified in the superior colliculus (SC) following injections of Phaseolus vulgaris-Leucoagglutinin in the ventral prepositus hypoglossi nucleus (PH) and adjacent reticular formation. Labelled axons distribute terminals within the intermediate and the deep layers on both sides. Within the former, their distribution reproduces the representation of the visual hemifield on the same side as the injected PH. Additionally the density of boutons gradually increases in the contralateral SC, from the projection of the area centralis towards the periphery, i.e. towards regions coding for larger saccades. Such a differential synaptic input may provide the neuronal basis for a temporal to spatial transformation of the feedback signal controlling gaze shifts. A theoretical model is proposed.

To determine how often Bunina bodies (BBs) appear in the medullary reticular formation (MRF) in amyotrophic lateral sclerosis (ALS), we microscopically examined 20 serial sections of MRFs from each of nine autopsied ALS cases, which had BBs in the lower motor neurons, including those of cranial motor nuclei. In 1 of them, the pontine tegmentum was examined in the same way. In 8 cases one to several BB-containing neurons in the MRF were seen. The case in which the pontine tegmentum was also investigated exhibited several neurons with BBs in this region. Some of the BBs in the MRFs were confirmed by electron microscopy. Thus, this study demonstrates the common appearance of BBs, although the number is small, in the MRF, indicating that pathological processes that undermine the lower motor neurons in ALS in some way also affect neurons other than motor neurons in this condition.

Physiological measurements suggest that the inhibition of primate spinothalamic tract cells by serotonin is mediated by myelinated axons. Previous morphologic studies emphasize that most serotonin-containing axons in the spinal cord are unmyelinated. Accordingly, the possibility that some serotonin-containing axons in the primate dorsolateral funiculus of the spinal cord are myelinated was investigated. Macaque monkeys were given L-tryptophan and the monoamine oxidase inhibitor, nialamide, intraperitoneally 1 h prior to sacrifice to increase axonal stores of serotonin. The animals were perfused (0.05 or 0.5% glutaraldehyde, 4% paraformaldehyde), and transverse sections of the thoracic cord were reacted with antibody against serotonin and then prepared for electron microscopy. Many of the immunostained axons in the dorsolateral funiculus included fine, myelinated fibers with diameters of 0.7-2.2 microns. Unmyelinated serotonin-containing axons were also observed. The observation of myelinated serotonin-containing axons in the white matter of the monkey dorsolateral funiculus contradicts the view that the descending serotoninergic projection consists entirely of unmyelinated fibers, particularly since the conduction velocity of the fine fibers would be too slow to account for the earliest latency of descending inhibition following stimulation in the brainstem. The presence of myelinated serotoninergic axons presumably accounts for the latencies reported for the inhibition of primate spinothalamic cells following stimulation of the periaqueductal gray, an inhibition that can be blocked with serotonin antagonists and that is associated with the release of serotonin in the dorsal horn.

Fibrous components of the connective tissue are light-microscopically classified into three types: collagen fibers, reticular fibers and elastic fibers. The present paper reviews the three-dimensional ultrastructure of these fibrous components, mainly based on our studies by scanning electron microscopy. The collagen fibers are shaped like tapes or cords about 1 to 20 microns in diameter. Each fiber is a bundle of fibrils running roughly parallel to each other. These collagen fibrils vary in diameter from 30 to 300 nm depending on their locating area of the body, and show a repeating pattern of depressed and protruding segments on the surface. The reticular fibers consist of collagen fibrils about 20-40 nm in diameter, which run singly or in small bundles. They are usually interwoven elaborately to form thin lace-like sheets or sheaths attaching to basal laminae of such cells as epithelial, endothelial and muscular cells. These fibers are considered to play an important role not only in adhering the cells to the collagen fibers, but also in constituting the skeletal framework suitable for individual cells and tissues. The elastic fibers consist of two different components: elastin and fibrillin. Elastin forms unit fibrils of 0.1-0.2 micron thickness which are arranged in bundles or laminae specific to individual organs and tissues. Fibrillin, on the other hand, forms microfibrils about 10 nm in diameter running in or along elastin bundles. These microfibrils also form delicate networks separate from elastin components. For a comprehensive understanding of the fibrous components in the connective tissue, the author proposed categorizing them into two systems: the collagen fibrillar system as a supporting framework of tissues and cells, and the fibrillin-elastin fibrillar system for distributing stressing forces uniformly in tissues.

We describe a modified technique for percutaneous denervation of the thoracic sympathetic chain by laser to treat selected cases of sympathetic causalgia of the upper extremities. The technique involves transpleural ablation with laser under thoracoscopic guidance through the second or third intercostal space-anterior axillary line. We also compare four different modalities of endoscopic denervation: A xenon chloride excimer laser (308 nm, 35 mJ/pulse, 20 pulses/sec, 2.2 mm catheter tip), CO2 laser (14 W, CW, 2 mm spot size), Nd:YAG laser (88 W, CW, 3 mm spot size), and radiofrequency-generated thermocoagulation (3 W, CW, 2.1 mm catheter tip) by performing bilateral thoracic sympathectomy on 12 mongrel dogs (three dogs each). Criteria analyzed included duration of exposure, power density, total energy output, laser penetration and spread, gross morphology, and scanning electron microscopy (SEM) of the destroyed neural tissue. Total ablation of the inferior segment of the stellate ganglion and the T1-T2 nerve roots by excimer laser required 83 +/1 1 Joules over an exposure period of 118 seconds. Ablation by CO2 and Nd:YAG laser required 153 +/- 13 Joules and 554 +/- 47 Joules delivered over 11 and 6 seconds respectively. In contrast, ablation of the same volume of nerve tissue by RF required 810 +/- 50 Joules over 270 seconds. SEM evaluation revealed that excimer and CO2 laser lesions were narrower in configuration compared to RF and Nd:YAG lesions which showed more lateral spread. The actual depth of penetration per 1 second exposure was similar for Excimer and CO2 (1.5 mm) and RF (1.3 mm), but deeper for Nd:YAG (3 mm).(ABSTRACT TRUNCATED AT 250 WORDS)