Small-diameter Dendrites Research Articles

Immunocytochemical and ultrastructural organization of the taste thalamus of the tree shrew (Tupaia belangeri).

Ventroposterior medialis parvocellularis (VPMP) nucleus of the primate thalamus receives direct input from the nucleus of the solitary tract, whereas the homologous thalamic structure in the rodent does not. To reveal whether the synaptic circuitries in these nuclei lend evidence for conservation of design principles in the taste thalamus across species or across sensory thalamus in general, we characterized the ultrastructural and molecular properties of the VPMP in a close relative of primates, the tree shrew (Tupaia belangeri), and compared these to known properties of the taste thalamus in rodent, and the visual thalamus in mammals. Electron microscopy analysis to categorize the synaptic inputs in the VPMP revealed that the largest-size terminals contained many vesicles and formed large synaptic zones with thick postsynaptic density on multiple, medium-caliber dendrite segments. Some formed triads within glomerular arrangements. Smaller-sized terminals contained dark mitochondria; most formed a single asymmetric or symmetric synapse on small-diameter dendrites. Immuno-EM experiments revealed that the large-size terminals contained VGLUT2, whereas the small-size terminal populations contained VGLUT1 or ChAT. These findings provide evidence that the morphological and molecular characteristics of synaptic circuitry in the tree shrew VPMP are similar to that in nonchemical sensory thalamic nuclei. Furthermore, the results indicate that all primary sensory nuclei of the thalamus in higher mammals share a structural template for processing thalamocortical sensory information. In contrast, substantial morphological and molecular differences in rodent versus tree shrew taste nuclei suggest a fundamental divergence in cellular processing mechanisms of taste input in these two species.

The Mouse Pulvinar Nucleus Links the Lateral Extrastriate Cortex, Striatum, and Amygdala.

The pulvinar nucleus is a large thalamic structure involved in the integration of visual and motor signals. The pulvinar forms extensive connections with striate and extrastriate cortical areas, but the impact of these connections on cortical circuits has not previously been directly tested. Using a variety of anatomical, optogenetic, and in vitro physiological techniques in male and female mice, we show that pulvinocortical terminals are densely distributed in the extrastriate cortex where they form synaptic connections with spines and small-diameter dendrites. Optogenetic activation of these synapses in vitro evoked large excitatory postsynaptic responses in the majority of pyramidal cells, spiny stellate cells, and interneurons within the extrastriate cortex. However, specificity in pulvinar targeting was revealed when recordings were targeted to projection neuron subtypes. The neurons most responsive to pulvinar input were those that project to the striatum and amygdala (76% responsive) or V1 (55%), whereas neurons that project to the superior colliculus were rarely responsive (6%). Because the pulvinar also projects directly to the striatum and amygdala, these results establish the pulvinar nucleus as a hub linking the visual cortex with subcortical regions involved in the initiation and control of movement. We suggest that these circuits may be particularly important for coordinating body movements and visual perception.SIGNIFICANCE STATEMENT We found that the pulvinar nucleus can strongly influence extrastriate cortical circuits and exerts a particularly strong impact on the activity of extrastriate neurons that project to the striatum and amygdala. Our results suggest that the conventional hierarchical view of visual cortical processing may not apply to the mouse visual cortex. Instead, our results establish the pulvinar nucleus as a hub linking the visual cortex with subcortical regions involved in the initiation and control of movement, and predict that the execution of visually guided movements relies on this network.

Enriched Expression of Neutral Sphingomyelinase 2 in the Striatum is Essential for Regulation of Lipid Raft Content and Motor Coordination

Sphingomyelinases are a family of enzymes that hydrolyze sphingomyelin to generate phosphocholine and ceramide. The brain distribution and function of neutral sphingomyelinase 2 (nSMase2) were elucidated in this study. nSMase2 mRNA expression was greatest in the striatum, followed by the prefrontal cortex, hippocampus, cerebellum, thalamus, brainstem, and olfactory bulb. The striatum had the highest level of nSMase2 protein expression, followed by the prefrontal cortex, thalamus, hippocampus, brainstem, and cerebellum. Dense immunolabeling was observed in the striatum, including the caudate-putamen, while moderately dense staining was found in the olfactory bulb and cerebral neocortex. Electron microscopy of the caudate-putamen showed nSMase2 immunoreaction product was present in small diameter dendrites or dendritic spines, that formed asymmetrical synapses with unlabeled axon terminals containing small round vesicles; and characteristics of glutamatergic axons. Lipidomic analysis of the striatum showed increase in long chain sphingomyelins, SM36:1 and SM38:1 after inhibition of nSMase activity. Quantitative proteomic analysis of striatal lipid raft fraction showed many proteins were downregulated by more than 2-fold after inhibition or antisense knockdown of nSMase; consistent with the notion that nSMase2 activity is important for aggregation or clustering of proteins in lipid rafts. Inhibition or antisense knockdown of nSMase2 in the caudate-putamen resulted in motor deficits in the rotarod and narrow beam tests; as well as decreased acoustic startle and improved prepulse inhibition of the startle reflex. Together, results indicate an important function of nSMase2 in the striatum.

Distribution and density of contacts from noradrenergic and serotonergic boutons on the dendrites of neck flexor motoneurons in the adult cat.

Serotonergic (5-HT) and noradrenergic (NA) input to spinal motoneurons is essential for generating plateau potentials and self-sustained discharges. Extensor motoneurons are densely innervated by 5-HT and NA synapses and have robust plateau potentials and self-sustained discharges. Conversely, plateau potentials and self-sustained discharges are very rare in flexor motoneurons. The most likely reasons for this difference are that flexor motoneurons have few 5-HT and NA synapses and/or they are distributed distant to the channels responsible for plateau potentials and self-sustained discharges. However, the distribution of 5-HT and NA synapses on flexor motoneurons is unknown. Here we describe the distribution and density of 5-HT and NA synapses on motoneurons that innervate the flexor neck muscle, rectus capitis anterior (RCA), in the adult cat. Using a combination of intracellular staining, fluorescent immunohistochemistry, and 3D reconstruction techniques, we found that 5-HT and NA synapses are widely distributed throughout the dendritic trees of RCA motoneurons, albeit with a strong bias to small-diameter dendrites and to medial dendrites in the case of NA contacts. The number of 5-HT and NA contacts per motoneuron ranged, respectively, from 381 to 1,430 and from 642 to 1,382, which is 2.3- and 1.4-fold less than neck extensor motoneurons (Montague et al., J Comp Neurol 2013;521:638-656). These results suggest that 5-HT and NA synapses on flexor motoneurons may provide a powerful means of amplifying synaptic currents without incurring plateau potentials or self-sustained discharges. This feature is well suited to meet the biomechanical demands imposed on flexor muscles during different motor tasks.

Nonuniform distribution of contacts from noradrenergic and serotonergic boutons on the dendrites of cat splenius motoneurons

The input-output properties of motoneurons are dynamically regulated. This regulation depends, in part, on the relative location of excitatory and inhibitory synapses, voltage-dependent and -independent channels, and neuromodulatory synapses on the dendritic tree. The goal of the present study was to quantify the number and distribution of synapses from two powerful neuromodulatory systems that originate from noradrenergic (NA) and serotonergic (5-HT) neurons. Here we show that the dendritic trees of motoneurons innervating a dorsal neck extensor muscle, splenius, in the adult cat are densely, but not uniformly innervated by both NA and 5-HT boutons. Identified splenius motoneurons were intracellularly stained with Neurobiotin. Using 3D reconstruction techniques we mapped the distributions of contacts formed by NA and 5-HT boutons on the reconstructed dendritic trees of these motoneurons. Splenius motoneurons received an average of 1,230 NA contacts (range = 647-1,507) and 1,582 5-HT contacts (range = 1,234-2,143). The densities of these contacts were 10 (NA) to 6 (5-HT)-fold higher on small compared to large-diameter dendrites. This relationship largely accounts for the bias of NA and 5-HT contacts on distal dendrites and is partially responsible for the higher density of NA contacts on dendrites located more than 200 μm dorsal to the soma. These results suggest that the neuromodulatory actions of NA and 5-HT are compartmentalized and regulate the input-output properties of motoneurons according to precisely arranged interactions with voltage-dependent and -independent channels that are primarily located on small-diameter dendrites.

N‐methyl D‐aspartate (NMDA) receptors endow dendrites of gonadotropin releasing‐hormone (GnRH) neurons with additional active properties

Dendrites of GnRH neurons express tetrodotoxin‐sensitive voltage‐gated sodium channels and generate action potentials. Other dendrites exhibiting action potentials possess additional active properties such as NMDA‐mediated potentials. We tested the hypothesis that GnRH dendrites express R1 subunits of NMDA receptors and exhibit amplification of sub‐threshold, excitatory post‐synaptic potentials (EPSPs) via NMDA receptor activation. Immunochemistry indicated GnRH dendrites express R1 subunits. Stimulating living GnRH dendrites with simulated sub‐threshold EPSPs resulted in plateau potentials and individual action potentials. When GABAergic inputs were blocked with picrotoxin (100 μM), repetitive action potentials occurred. The NMDA receptor antagonist D‐2‐amino‐5‐phosphono‐valeric (50 μM) eliminated responses. In small diameter dendrites, NMDA receptors would have a pronounced impact. Activating simulated NMDA conductances (at 560 μm dendrite length) in our compartmental models of GnRH neurons resulted in repetitive action potentials. Activating proximal NMDA receptors (at 200 and 100 μm dendrite length) diminished the impact of NMDA receptors. Our findings indicate GnRH dendrites exhibit regionalized expression of the NMDA conductance thereby forming location‐dependent microdomains where estrogen‐sensitive GABAergic neurons can regulate local excitability.

Expression profile of multiple secretory phospholipase A 2 isoforms in the rat CNS: Enriched expression of sPLA 2-IIA in brainstem and spinal cord

Phospholipases A 2 (PLA 2) are enzymes which cleave the sn-2 ester bond in membrane phospholipids to release free fatty acids and lysophospholipids. The present study aimed to elucidate the expression profile of multiple secretory phospholipase A 2 (sPLA 2) isoforms in the normal rat CNS with focus on sPLA 2-IIA in the brainstem and spinal cord. Quantitative RT-PCR analysis showed that sPLA 2-IB expression was low throughout the CNS, sPLA 2-IIA expression was high in the brainstem and spinal cord, sPLA 2-IIC expression was high in the cerebral neocortex, hippocampus and thalamus/hypothalamus, sPLA 2-V expression was high in the olfactory bulb and cerebellum, and sPLA 2-X was expressed at very low levels in the normal CNS. Of the isoforms, sPLA 2-IIA mRNA expression was highest in the brainstem and spinal cord suggesting that this could be the most relevant isoform in the ascending pain pathway. Western blot analysis showed high level of sPLA 2-IIA expression in the brainstem and cervical, thoracic and lumbar spinal segments but low level of expression in other parts of the brain. sPLA 2-IIA was localized by immunohistochemistry to the spinal trigeminal and facial motor nuclei and dorsal- and ventral-horns of the spinal cord. The enzyme was found on the endoplasmic reticulum of neuronal cell bodies and small diameter dendrites or dendritic spines at electron microscopy. The expression of sPLA 2-IIA in the dorsal horn and spinal trigeminal nucleus is consistent with previous results which showed an important role of CNS sPLA 2 in nociceptive transmission.

Subcellular distribution of low‐voltage activated T‐type Ca2+ channel subunits (Cav3.1 and Cav3.3) in reticular thalamic neurons of the cat

Low-voltage-activated (LVA) Ca(2+) channels play a critical role in the generation of burst firing in the thalamus. Recently, three LVA Ca(2+) channel isoforms (Ca(v)3.1, Ca(v)3.2, Ca(v)3.3) have been identified in the reticular thalamic nucleus (RE). Previous electrophysiological and modelling studies have suggested that kinetically different T-type channels might be expressed in a compartmentalized manner in RE cells. However, their precise subcellular distribution has not been fully elucidated. Using light and electron microscopic (EM) immunocytochemistry, we investigated the subcellular expression pattern of Ca(v)3.1 and Ca(v)3.3 channel subunits in RE neurons of the cat. Fluorescent and peroxidase labelling demonstrated the presence of Ca(v)3.1 channel predominantly on the somata and proximal dendrites and Ca(v)3.3 channels on cell bodies. Quantitative immunogold localization disclosed that Ca(v)3.1 and Ca(v)3.3 isoforms showed 5.8- and 8.7-fold higher density, respectively, in the cytoplasm compared with somatic plasma membrane. Density of Ca(v)3.1 isoform in the somatic plasma membrane was 2.21-fold higher compared with Ca(v)3.3 subunit. In the dendritic plasma membrane, Ca(v)3.1 channel isoform was expressed throughout the entire dendritic tree. In contrast, Ca(v)3.3 isoform was absent from large-caliber, presumably proximal dendritic segments. Quantitative comparison showed that the relative density of immunogold particles compared with dendritic surface was 8.9- and 14.8-fold higher for Ca(v)3.1 and Ca(v)3.3, respectively, in small-diameter dendrites than in large proximal dendritic segments or somata. Our results demonstrate a higher density of low-threshold Ca(2+) channels in distal dendrites and provide further evidence of the role of RE neuron dendrites in the generation of prolonged, low-threshold spike bursts.

A simple method for quantitative calcium imaging in unperturbed developing neurons

Calcium imaging has been widely used to address questions of neuronal function and development. To gain deeper insights into the actions of calcium as a second messenger, but also to measure synaptic function, it is necessary to quantify the level of calcium at rest and during calcium transients. While quantification of calcium levels is straightforward when using ratiometric calcium indicators, these dyes have several draw-backs due to their short wavelength excitation spectra, such as light scattering and cytotoxicity. In contrast, many single-wavelength indicators exhibit superior photostability, low phototoxicity, extended dynamic ranges and very high signal to noise ratios. However, quantifying calcium levels in unperturbed neurons has not been performed with these indicators. Here, we explore a new approach for determining the calcium concentration at rest as well as calcium rises during evoked and spontaneous neuronal activity in unperturbed developing neurons using a single-wavelength calcium indicator. We show that measuring the maximal fluorescence at the end of an imaging experiment allows determining calcium levels with high resolution. Specifically, we assessed the limits of calcium measurements with a CCD camera in small neuronal processes and found that even in small diameter dendrites and spines the intracellular calcium concentration and its changes can be estimated accurately. This approach may not only allow mapping patterns of neuronal activity quantitatively with the resolution of single synapses and a few tens of milliseconds, but also facilitate investigating the role of calcium as a second messenger.

3D electron microscopic reconstruction of segments of rat cerebellar purkinje cell dendrites receiving ascending and parallel fiber granule cell synaptic inputs

Growing physiological evidence suggests that there are functional differences between synapses made by the ascending and parallel fiber segments of the granule axon on cerebellar Purkinje cells. Supporting this view, our previous electron microscopic studies suggested that these synapses also contacted different regions of the Purkinje cell dendrite, and in particular that ascending segment synapses are made exclusively on the smallest diameter Purkinje cell dendrites. In the current study we used serial electron microscopic techniques to reconstruct Purkinje cell dendritic segments up to almost 10 mum in length. Using a combination of anatomical and immunological labeling techniques we identified the ascending or parallel fiber origins of the excitatory synaptic inputs onto dendritic spines, as well as the location of inhibitory synapses made directly on the dendritic shaft. The results confirmed that there are regions of the Purkinje cell dendrite receiving exclusively ascending or parallel fiber synapses and that ascending segment synapses are only found on small-diameter dendrites. In addition, we describe for the first time small-diameter dendritic regions contacted by both types of excitatory synapses. While our data suggest that the majority of inhibitory inputs to the Purkinje cell tree are associated with parallel fiber synaptic inputs, we also found inhibitory inputs on dendritic regions with mixed ascending and parallel fiber inputs, or exclusively parallel fiber inputs. The finding that ascending and parallel fiber inputs can be segregated on the Purkinje cell dendritic tree provides further evidence that these excitatory granule cell synaptic inputs may be functionally distinct.

Calcium Spikes in Basal Dendrites of Layer 5 Pyramidal Neurons during Action Potential Bursts

Patch-clamp recording from dendrites has lead to a significant increase in our understanding of the mechanisms underlying signal integration and propagation in neurons. The majority of synaptic input to neurons, however, is made onto small-diameter dendrites, currently beyond the scope of patch-clamp recording techniques. Here we use both calcium and voltage imaging to investigate propagation of action potentials (APs) in fine basal dendrites of cortical layer 5 pyramidal neurons. High-frequency (200 Hz) AP bursts caused supralinear increases in dendritic calcium at distal, but not proximal, basal locations. Supralinear increases in dendritic calcium were also observed at distal basal locations during AP trains above a critical frequency (approximately 100 Hz). Using voltage imaging, we show that single APs undergo significant attenuation as they propagate into basal dendrites, whereas AP bursts lead to generation of dendritic calcium spikes. Focal and bath application of 4-AP increased the amplitude of calcium transients evoked by APs at distal, but not proximal, locations, suggesting that A-type potassium channels regulate AP backpropagation into basal dendrites. Finally, we show that pairing EPSPs with AP bursts is an effective means of activating synaptic NMDA receptors in basal dendrites. The experimental observations on the role of A-type potassium channels in regulation of AP backpropagation in basal dendrites, as well as the generation of dendritic calcium spikes during AP bursts, were reproduced in a morphologically realistic neuronal model with uniform distributions of dendritic sodium, calcium, and potassium channels. Together, these findings have important implications for understanding dendritic integration and synaptic plasticity in cortical basal dendrites.

Ultrastructure and synaptology of the paratrigeminal nucleus in the rat: Primary pharyngeal and laryngeal afferent projections

The paratrigeminal nucleus (PTN) receives primary afferent projections from the aerodigestive tract and orofacial regions and plays a role in the integration of visceral and somatic information. This study describes the fine structure of the rat PTN and the synaptology of primary afferent projections from the pharynx and larynx. Injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or cholera toxin-HRP (CT-HRP) were made into the wall of the pharynx or larynx to label primary afferent axon terminals. Light microscopic observations demonstrated that afferent axons terminated bilaterally in overlapping fields in the PTN. Electron microscopic observations of the PTN revealed that there were three distinct classes of neurons, based on morphology and axosomatic contacts. The most abundant neurons, Type 1, were fusiform in shape and received very few or no axosomatic contacts. Type 2 neurons contained prominent Nissl substance (rough endoplasmic reticulum) and few axosomatic contacts, while Type 3 neurons had many axosomatic synapses. Terminals containing round, clear vesicles and forming asymmetric contacts (round asymmetric, RA) with dendrites were the predominant synaptic type in the PTN. Primary afferent terminals from the pharynx and larynx were of the RA type and formed synaptic contacts with small-diameter (<1 microm) dendrites. Visceral primary afferent inputs from the pharynx and larynx overlap with trigeminal somatic afferents in the PTN and have similar synaptic morphology. The results support the concept that the PTN provides an anatomical substrate for mediating viscerovisceral and somatovisceral reflexes via efferent connections with autonomic centers in the brainstem.

Projections from the cat posterior lateral hypothalamus to the ventral part of the oral pontine reticular nucleus contain a GABAergic component

The posterior lateral hypothalamus (PLH) has long been considered crucial to normal wakefulness while the ventral part of the oral pontine reticular nucleus (vRPO) is involved in the generation and maintenance of rapid eye movement (REM) sleep. However, to date, there is no information on the ultrastructure or neurotransmitter content of the hypothalamo-reticular projection. In the present study, we examined the morphology and synaptic organization of PLH terminals in the vRPO using PLH injections of biotinylated dextran amine (BDA) as well as of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Since some PLH neurons are GABAergic, we used a post-embedding immunogold technique to determine whether any anterogradely labeled terminals were GABA-immunopositive. Electron microscope analyses revealed a variety of ultrastructural features in the vRPO anterogradely labeled terminals. Although most labeled terminals (over 63%) formed symmetric synapses on vRPO somata and dendrites, others made asymmetric synapses on vRPO dendrites. The relative percentages of labeled terminals observed on large, medium and small diameter dendrites were 44.3±5.5%, 35.3±3.0% and 20.4±3.1%, respectively. Finally, post-embedding immunogold technique revealed that there are GABA-immunopositive and immunonegative components to this projection, indicating that GABA is one of the transmitters used by the PLH cells that project to the vRPO. Furthermore, most, if not all, of the GABA-labeled axon terminals formed symmetric synapsis. In conclusion, our results suggest that the PLH could modulate the physiological responses of vRPO neurons through a GABAergic pathway as well as by other inhibitory and/or excitatory pathways. Activation of the descending PLH GABAergic projection may inhibit the REM sleep-inducing neurons within the vRPO and thus contribute to the suppression of REM sleep activation during wakefulness.

A light and electron microscopic study of betaine/GABA transporter distribution in the monkey cerebral neocortex and hippocampus.

The present study aimed to elucidate the distribution of betaine/gamma-aminobutyric acid (GABA) transporter-1 (BGT-1) in the normal monkey cerebral neocortex and hippocampus by immunoperoxidase and Immunogold labelling. BGT-1 was observed in pyramidal neurons in the cerebral neocortex and the CA fields of the hippocampus. Large numbers of small diameter dendrites or dendritic spines were observed in the neuropil. These made asymmetrical synaptic contacts with unlabelled axon terminals containing small round vesicles, characteristic of glutamatergic terminals. BGT-1 label was observed in an extra-perisynaptic region, away from the post-synaptic density. Immunoreactivity was not observed in portions of dendrites that formed symmetrical synapses, axon terminals, or glial cells. The distribution of BGT-1 on dendritic spines, rather than at GABAergic axon terminals, suggests that the transporter is unlikely to play a major role in terminating the action of GABA at a synapse. Instead, the osmolyte betaine is more likely to be the physiological substrate of BGT-1 in the brain, and the presence of the transporter in pyramidal neurons suggests that these neurons utilize betaine to maintain osmolarity.

Reconstruction of motoneuronal morphology using two-photon molecular excitation microscopy

Neuronal somato-dendritic morphology is an important determinant of the conduction properties of neurons, and thus the way neurons process information. Current semi-automated methods of neuronal tracing by wide-field light microscopy of diaminobenzidine (DAB) labelled neurons suffer a number of limitations. Firstly, the required fixation introduces a variable spatial distortion. Secondly, under optimal conditions the spatial resolution is at best diffraction limited and these conditions are rarely achieved. Current estimates of dendritic diameter indicate a lower limit of ~0.5 μm [1]. This is close to the limit of resolution, and the diameter of smaller dendrites may therefore have been overestimated. Thirdly, the requirement for extensive, time-consuming user-input limits the practicality of the procedure. In this study, two-photon molecular excitation microscopy (2PM) was used to reconstruct the morphology of living, cytoplasmically-labelled motoneurons (MNs) from the hypoglossal (XII) nucleus of neonatal mice. Using the improved imaging capabilities of 2PM in light-scattering tissues and combining it with advanced image analysis we overcome the limitations of conventional light microscopy described above. MNs in isolated transverse brainstem slices containing the XII nucleus [2] were visualised using infra-red differential contrast microscopy. Selected MNs were whole-cell voltage-clamped and fluorescent dyes iontophoresed via the patch-pipette. Slices were then transferred to an inverted confocal microscope modified for 2PM, and 3D stacks of images were acquired at diffraction-limited resolution. Custom-written analysis routines were used to assess dendrite diameters at selected points. Fluorescence intensity was measured along a line orthogonal to the dendrite at these points and diameter defined as full width at half maximum intensity. While the resolution of this method is also limited by the wavelength of light, we overcame this limitation by using the fact that in 2PM the signal from structures smaller than the resolution limit (0.4 μm in x-y, 0.8 μm in z) is proportional to the illuminated volume. It is therefore possible to relate the recorded fluorescence intensity to the local diameter of sub resolution dendrites if they are modelled as cylinders [3]. By combining this approach with fully automatic tracing we can quantify the morphology and local diameters of intact functioning MNs and unequivocally clarify the existence of small diameter (<0.5 μm) den-drites. To date, reconstructions have been completed for the proximal regions of the somato-dendritic tree (<250 μm from the soma). These are comparable in branching pattern, complexity and local diameters (0.8–4 μm) to reconstructions of XII MNs made using wide-field microscopy [1]. Significant progress has also being made towards completion of a fast, robust, fully automatic system for tracing and quantifying the morphology of whole neurons from 2PM volume data. The functional significance of somato-dendritic complexity is a rapidly expanding research field. Accurate reconstructions of specific somato-dendritic morphologies, and subsequent identification of the generalised principles describing dendritic morphology, is essential to developing models of neuronal behaviour and to elucidating the roles neurons play in information processing.

A light and electron microscopic study of glutamate receptors in the monkey subthalamic nucleus.

The distribution of glutamate receptors in the monkey subthalamic nucleus was studied using affinity purified polyclonal antibodies to GluR1, phosphorylated GluR1, GluR2/3, NMDAR1, mGluR1a and mGluR5. Intense staining for both the unphosphorylated and the phosphorylated forms of the AMPA receptor subunit GluR1 was observed in the cell bodies and proximal dendrites of neurons in this nucleus. In comparison to GluR1, less intense staining for GluR2/3 was observed in the cell bodies and processes. NMDAR1 immunoreactivity was present in cell bodies and large numbers of small diameter dendrites. Light staining was observed in cell bodies with mGluR1a and no staining was observed on cell bodies with mGluR5. The neuropil, however, contained many processes that were labeled for mGluR1a or mGluR5. Electron microscopy showed that label was present in cytoplasmic locations in cell bodies and dendrites, in addition to components of the synaptic region, in sections stained for GluR1, GluR2/3 and NMDAR1. In contrast, very lightly labeled or unlabeled cell bodies but labeled dendrites and axon terminals, was observed in sections stained for mGluR1a and mGluR5. In addition to neural processes, occasional astrocytic processes were also labeled for mGluR5. Of the immunogold particles that were associated with components of the synaptic region, label for ionotropic glutamate receptors was mostly present on postsynaptic densities, whilst that for metabotropic glutamate receptors was mostly present in a perisynaptic location. The ratio of GluR1/GluR2 messenger RNAs has been reported to increase in the aged hippocampus (PAGLIUSI, S. R., GERRARD, P., ABDALLAH, M., TALABOT, D. & CATSICAS, S. (1994) Neuroscience 61, 429-433.), and it is possible that a similar change in the ratio of GluR1 and GluR2 may occur in neurons of the subthalamic nucleus with age. It is postulated that this could result an increase in calcium permeability via AMPA receptors, and an enhancement of excitatory transmission in this nucleus.

Ultrastructural synaptic organization of axon terminals in the ventral part of the cat oral pontine reticular nucleus.

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.

Central distribution of synaptic contacts of primary and secondary jaw muscle spindle afferents in the trigeminal motor nucleus of the cat

Little is known about the differences of the terminations of group Ia and group II afferents within the brainstem or spinal cord. The present study was performed to classify cat jaw muscle spindle afferents by the use of succinylcholine (SCh) and to examine the morphological characteristics of the physiologically classified afferents at the light and electron microscopic levels through the use of the intra-axonal horseradish peroxidase (HRP) injection technique. The effects of SCh on stretch responses of 119 jaw muscle spindle afferents from the masseter were examined. The SCh converted the single skew distribution of the values for dynamic index (DI) into a bimodal one. Fifty-eight and 61 afferents were classified as group Ia and group II afferents, respectively. The central projections of 17 intra-axonally stained afferents (10 group Ia and 7 group II afferents) were examined. The spindle afferents terminated mainly in the supratrigeminal nucleus (Vsup), region h, and the dorsolateral subdivision of trigeminal motor nucleus (Vmo.dl) but differed in the pattern of projections of group Ia and group II afferents. The proportion of group Ia afferent terminals was higher in Vmo.dl but lower in Vsup than that of group II afferents. In Vmo.dl, the proportion of group Ia afferent terminals was higher in the central region but lower in the more outer regions than that of group II afferents. The ultrastructure of serially sectioned afferent boutons (63 group Ia and 72 group II boutons) also was examined. The boutons from the two groups were distributed widely from the soma to small-diameter dendrites, but the frequency of synaptic contacts on proximal dendrites was higher in group Ia than group II afferents. The present study provides evidence that the two groups of jaw muscle spindle afferents differ in their central projection and the spatial distribution of their synaptic contacts on Vmo.dl neurons. J. Comp. Neurol. 391:50–63, 1998. © 1998 Wiley-Liss, Inc.

Central distribution of synaptic contacts of primary and secondary jaw muscle spindle afferents in the trigeminal motor nucleus of the cat

Little is known about the differences of the terminations of group Ia and group II afferents within the brainstem or spinal cord. The present study was performed to classify cat jaw muscle spindle afferents by the use of succinylcholine (SCh) and to examine the morphological characteristics of the physiologically classified afferents at the light and electron microscopic levels through the use of the intra-axonal horseradish peroxidase (HRP) injection technique. The effects of SCh on stretch responses of 119 jaw muscle spindle afferents from the masseter were examined. The SCh converted the single skew distribution of the values for dynamic index (DI) into a bimodal one. Fifty-eight and 61 afferents were classified as group Ia and group II afferents, respectively. The central projections of 17 intra-axonally stained afferents (10 group Ia and 7 group II afferents) were examined. The spindle afferents terminated mainly in the supratrigeminal nucleus (Vsup), region h, and the dorsolateral subdivision of trigeminal motor nucleus (Vmo.dl) but differed in the pattern of projections of group Ia and group II afferents. The proportion of group Ia afferent terminals was higher in Vmo.dl but lower in Vsup than that of group II afferents. In Vmo.dl, the proportion of group Ia afferent terminals was higher in the central region but lower in the more outer regions than that of group II afferents. The ultrastructure of serially sectioned afferent boutons (63 group Ia and 72 group II boutons) also was examined. The boutons from the two groups were distributed widely from the soma to small-diameter dendrites, but the frequency of synaptic contacts on proximal dendrites was higher in group Ia than group II afferents. The present study provides evidence that the two groups of jaw muscle spindle afferents differ in their central projection and the spatial distribution of their synaptic contacts on Vmo.dl neurons.

Ultrastructural observations of synaptic connections of vibrissa afferent terminals in cat principal sensory nucleus and morphometry of related synaptic elements.

Previous work suggests that slowly adapting (SA) periodontal afferents have different synaptic arrangements in the principal (Vp) and oral trigeminal nuclei and that the synaptic structure associated with transmitter release may be related directly to bouton size. The present study examined the ultrastructures of SA and fast adapting (FA) vibrissa afferents and their associated unlabeled axonal endings in the cat Vp by using intra-axonal labeling with horseradish peroxidase and a morphometric analysis. All SA and FA afferent boutons contained clear, round, synaptic vesicles. All the FA and most SA boutons were presynaptic to dendrites, but a few SA boutons were axosomatic. Both types of bouton were frequently postsynaptic to unlabeled axonal ending(s) containing pleomorphic, synaptic vesicles (P-ending). The size of labeled boutons was larger in FA than SA afferents, but the size of dendrites postsynaptic to labeled boutons was larger for SA than FA afferents. Large-sized FA and SA boutons made synaptic contacts with small-diameter dendrites. The size of FA and SA boutons was larger than that of their associated P-endings. A morphometric analysis made on the pooled data of SA and FA boutons indicated that apposed surface area, active zone number, total active zone area, vesicle number, and mitochondrial volume were highly correlated in a positive linear manner with labeled bouton volume. These relationships were also applicable to unlabeled P-endings, but the range of each parameter was smaller than that of the labeled boutons. These observations provide evidence that the two functionally distinct types of vibrissa afferent manifest unique differences but share certain structural features in the synaptic organization and that the ultrastructural "size principle" proposed by Pierce and Mendell ([1993] J. Neurosci. 13:4748-4763) for Ia-motoneuron synapses is applicable to the somatosensory system.