BDA-labeled Terminals Research Articles

Glutamatergic neurons in the lateral periaqueductal gray innervate neurokinin-1 receptor-expressing neurons in the ventrolateral medulla of the rat

The neural pathways underlying the respiratory responses elicited by electrical or chemical stimulation of the lateral part of the periaqueductal gray (lPAG) remain unsettled. In the present study, we examined the lPAG projection to neurokinin-1 receptor (NK1R)-immunoreactive (ir) neurons in the ventrolateral medulla (VLM) which have been implicated in the control of respiration. After biotinylated dextranamine (BDA) injection into the lPAG, NK1R-ir neurons in the rostral VLM were embedded in the plexus of BDA-labeled fibers. At the electron microscopic level, the BDA-labeled terminals made asymmetrical synaptic contacts predominantly with dendrites and additionally with somata of the NK1R-ir neurons. Using retrograde tracing combined with in situ hybridization, we demonstrated that the vast majority of the lPAG neurons projecting to the rostral VLM were positive for vesicular glutamate transporter 2 (VGLUT2) mRNA, but not for glutamic acid decarboxylase 67 mRNA. Using a combination of anterograde tracing and immunohistochemistry, we further demonstrated that the lPAG axon terminals with VGLUT2 immunoreactivity made close apposition with the NK1R-ir neuronal profiles in the rostral VLM. These data suggest that lPAG neurons exert an excitatory influence on NK1R-expressing neurons in the rostral VLM for the control of respiration.

Topography of descending projections from anterior insular and medial prefrontal regions to the lateral habenula of the epithalamus in the rat

The epithalamic lateral nucleus of the habenula (LHb) plays a key role in regulating firing of dopamine and serotonin neurons in the midbrain and is thereby involved in various cognitive and affective behaviors. It is not yet clear, however, from where the LHb receives cognitive and affective information relevant to its regulation of the midbrain monoaminergic systems. The prefrontal cortex would be among the ideal sources. Here, using anterograde and retrograde tracer injections in the rat brain, we characterized the topography of the corticohabenular projections. Following injections of cholera toxin subunit B into the LHb, retrogradely labeled neurons were produced in the anterior insular, cingulate, prelimbic and infralimbic cortices. Consistent with this retrograde tracing, injections of biotinylated dextran amine (BDA) into these cortical regions labeled robust terminals in the LHb. Our quantification of the BDA-impregnated varicosities revealed that projections from the anterior insula terminated mainly in the intersection regions of the lateral and ventral two-thirds of the LHb, while projections from the cingulate cortex terminated mainly in the lateral two-thirds of the LHb. By comparison, BDA-labeled terminals originating from the medial prefrontal regions were contained mainly in the medial plus ventral one-third of LHb. Based on these data, we hypothesize that LHb provides a link for conveying cognitive and affective information from prefrontal and insular regions to the midbrain monoaminergic centers.

Neurochemical Properties of the Synapses in the Pathways of Orofacial Nociceptive Reflexes

The brainstem premotor neurons of the facial nucleus (VII) and hypoglossal (XII) nucleus can integrate orofacial nociceptive input from the caudal spinal trigeminal nucleus (Vc) and coordinate orofacial nociceptive reflex (ONR) responses. However, the synaptoarchitectures of the ONR pathways are still unknown. In the current study, we examined the distribution of GABAergic premotor neurons in the brainstem local ONR pathways, their connections with the Vc projections joining the brainstem ONR pathways and the neurochemical properties of these connections. Retrograde tracer fluoro-gold (FG) was injected into the VII or XII, and anterograde tracer biotinylated dextran amine (BDA) was injected into the Vc. Immunofluorescence histochemical labeling for inhibitory/excitatory neurotransmitters combined with BDA/FG tracing showed that GABAergic premotor neurons were mainly distributed bilaterally in the ponto-medullary reticular formation with an ipsilateral dominance. Some GABAergic premotor neurons made close appositions to the BDA-labeled fibers coming from the Vc, and these appostions were mainly distributed in the parvicellular reticular formation (PCRt), dorsal medullary reticular formation (MdD), and supratrigeminal nucleus (Vsup). We further examined the synaptic relationships between the Vc projecting fibers and premotor neurons in the VII or XII under the confocal laser-scanning microscope and electron microscope, and found that the BDA-labeled axonal terminals that made asymmetric synapses on premotor neurons showed vesicular glutamate transporter 2 (VGluT2) like immunoreactivity. These results indicate that the GABAergic premotor neurons receive excitatory neurotransmission from the Vc and may contribute to modulating the generation of the tonic ONR.

Glutamatergic Kölliker–Fuse nucleus neurons innervate hypoglossal motoneurons whose axons form the medial (protruder) branch of the hypoglossal nerve in the rat

This study was performed to understand the anatomical substrates for Kölliker–Fuse nucleus (KFN) modulation of respiratory-related tongue movement. After application of cholera toxin B subunit (CTb) to the medial branch of the hypoglossal nerve (HGn) and injection of biotinylated dextran amine (BDA) into the KFN ipsilaterally, an overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found in the ventral compartment of the hypoglossal nucleus (HGN) ipsilateral to the application and injection sites. At the electron microscopic level, the BDA-labeled terminals made asymmetrical synaptic contacts predominantly with dendrites of the HGN neurons, some of which were labeled with CTb. Using retrograde tracing combined with in situ hybridization, we demonstrated that almost all the KFN neurons sending their axons to the HGN were positive for vesicular glutamate transporter (VGLUT) 2 mRNA but not glutamic acid decarboxylase 67 mRNA. Using a combination of anterograde and retrograde tracing techniques and immunohistochemistry for VGLUT2, we further demonstrated that the KFN axon terminals with VGLUT2 immunoreactivity established close contact with the HGN motoneurons whose axons constitute the medial branch of the HGn. The present results suggest that glutamatergic KFN fibers may exert excitatory influence upon the HGN motoneurons sending their axons to the medial branch of the HGn for the control of protruder tongue muscles contraction to maintain airway patency during respiration.

Neuroanatomical and neurochemical organization of projections from the central amygdaloid nucleus to the nucleus retroambiguus via the periaqueductal gray in the rat

The periaqueductal gray (PAG)-nucleus retroambiguus (NRA) pathway has been known to be involved in the control of vocalization and sexual behavior. To know how the amygdaloid complex influences the PAG-NRA pathway, here we first examined the synaptic organization between the central amygdaloid nucleus (CeA) fibers and the PAG neurons that project to the NRA by using anterograde and retrograde tract-tracing techniques in the rat. After ipsilateral injections of biotinylated dextran amine (BDA) into the CeA and cholera toxin B subunit (CTb) into the NRA, the prominent overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found ipsilaterally in the lateral/ventrolateral PAG, where some of the BDA-labeled terminals made symmetrical synaptic contacts with somata and dendrites of the CTb-labeled neurons. After CTb injection into the lateral/ventrolateral PAG, CTb-labeled neurons were distributed mainly in the medial division of the CeA. After BDA injection into the lateral/ventrolateral PAG, BDA-labeled fibers were distributed mainly in and around the NRA within the medulla oblongata. Using a combined retrograde tracing and in situ hybridization technique, we further demonstrated that more than half of the CeA neurons labeled with Fluoro-Gold (FG) injected into the lateral/ventrolateral PAG were positive for glutamic acid decarboxylase 67 mRNA and that the vast majority of PAG neurons labeled with FG injected into the NRA expressed vesicular glutamate transporter 2 mRNA. The present results suggest that the glutamatergic PAG-NRA pathway is under the inhibitory influence of the GABAergic CeA neurons.

A possible monosynaptic pathway links the pedunculopontine tegmental nucleus to thalamostriatal neurons in the hooded rat

The thalamic lateral posterior nucleus (LP) of the hooded rat is regarded as a relay nucleus for the transmission of information from visuomotor-related structures such as the superior colliculus, pedunculopontine tegmental nucleus (PPT) and substantia nigra, pars reticulata, to visual cortical areas as well as the striatum. The aim of the present study was to examine the relationships of the thalamo-striatal projection neurons with the LP afferent fibers derived from the PPT, using injections of the anterograde tracer biotinylated dextran amine (BDA) and the retrograde tracer cholera toxin-B (CTB) into the PPT and the striatum, respectively. Findings showed that the location of terminals derived from PPT and LP neurons projecting to the striatum overlapped considerably in the ventral portion of the LP. Ultrastructural observations within this overlapped LP region showed that BDA-labeled terminals make synaptic contacts with dendrites of CTB-labeled neurons. The present results thus provide morphological support for the contention that information from the PPT neurons is relayed to the striatum through by the LP.

Innervation of orexin/hypocretin neurons by GABAergic, glutamatergic or cholinergic basal forebrain terminals evidenced by immunostaining for presynaptic vesicular transporter and postsynaptic scaffolding proteins.

Orexin/hypocretin (Orx) neurons are critical for the maintenance of waking in association with behavioral arousal and postural muscle tone, since with their loss narcolepsy with cataplexy occurs. Given that basal forebrain (BF) neurons project to the hypothalamus and play important diverse roles in sleep/wake states, we sought to determine whether acetylcholine (ACh), glutamate (Glu), and/or GABA-releasing BF neurons innervate and could thereby differentially regulate the Orx neurons. From discrete injections of biotinylated dextran amine (BDA, 10,000 MW) into the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) in the rat, BDA-labeled fibers projected to the lateral hypothalamus (LH), perifornical area (PF), and dorsomedial hypothalamus (DMH), where approximately 41%, approximately 11%, and 9% of Orx-positive (+) neurons were respectively contacted in each region. Employing triple fluorescent staining for Orx, BDA, and presynaptic vesicular (V) transporters (T), we found that only 4% of the innervated Orx+ neurons in the LH were contacted by BDA+[VAChT+] terminals, whereas approximately 31% and approximately 67% were respectively contacted by BDA+[VGluT2+] and BDA+[VGAT+] terminals. In 3D-rendered and rotated confocal images, we confirmed the latter contacts and examined staining for postsynaptic proteins PSD-95, a marker for glutamatergic synapses, and gephyrin, a marker for GABAergic synapses, that were located on Orx+ neurons facing BDA-labeled terminals in approximately 20% and approximately 50% of contacts, respectively. With such synaptic input, BF glutamatergic neurons can excite Orx neurons and thus act to maintain behavioral arousal with muscle tone, whereas GABAergic neurons can inhibit Orx neurons and thus promote behavioral quiescence and sleep along with muscle atonia.

Hypoxia–sensitive glutamatergic neurons of solitary tract nucleus (NTS) innervate the retrotrapezoid nucleus (RTN) in rats

Central chemoreceptor neurons reside within the RTN. In this study, we tested whether RTN receives excitatory inputs from hypoxia-sensitive neurons located in commissuralis NTS (commNTS). After iontophoretic injection of the anterograde tracer biotinylated dextran amine (BDA) in commNTS, most (53%) BDA-labeled terminals in the RTN were immunoreactive for vesicular glutamate transporter-2 (VGLUT2) but very few (5%) were immunoreactive for glutamic acid decarboxylase (GAD67). Awake rats were exposed to hypoxia (n=6) or normoxia (control) (n=5) one week after iontophoretic injection of the retrograde tracer cholera toxin B (CTB) into the RTN. Hypoxia-activated neurons were identified by the presence of Fos-immunoreactive (Fos-ir) nuclei. Fos-ir commNTS neurons retrogradely labeled with CTB were detected only in hypoxia-treated rats. In commNTS, 51% of Fos-ir neurons with projections to RTN (CTB- immunoreactive) were glutamatergic and 2% were GABAergic. In conclusion, RTN chemoreceptors may receive an excitatory glutamatergic input from peripheral chemoreceptors via a single relay in commNTS. Supported by: HL 74011 (PGG); Capes-BEX 3495/04-3 (TSM).

Bilateral corticospinal projections arise from each motor cortex in the macaque monkey: a quantitative study.

The corticospinal projection is considered to influence fine motor function through nearly exclusively contralateral projections from the cortex in primates. However, unilateral lesions to this system in various species are frequently followed by significant functional improvement, raising the possibility that bilateral projections of this pathway may exist or emerge after injury. To examine the detailed anatomy and projections of the corticospinal motor neurons in rhesus monkeys (n = 4), we injected the high-resolution anterograde tracer biotinylated dextran amine (BDA) into 126 sites centered about the right lower extremity (LE) primary motor cortex. Projection and termination patterns were quantified at lumbar levels L1, L4, and L7 and mapped by using serial-section reconstructions. Notably, a mean of 10.1 +/- 0.6% (+/- SEM) of corticospinal tract (CST) axons descended in the lateral CST ipsilateral to the cortical BDA injection, and 87.9 +/- 1.0% of total CST axons projected in the contralateral lateral CST. The ipsilateral ventral CST contained only 1.0 +/- 0% of all projecting CST axons, whereas the contralateral ventral CST contained 0.3 +/- 0.2% of all axons. In addition, a minor dorsal column CST projection was identified. Measurement of BDA-labeled terminals in the spinal cord gray matter revealed that 11.2 +/- 2.2% of CST axons terminated ipsilateral to the side of cortical injection, and the remainder terminated contralaterally. As previously reported, most CST axons terminated in spinal cord laminae V-VIII, as well as the laterodorsal motoneuronal group of lamina IX (which innervates distal extremity muscles). Notably, many CST axons crossed the spinal cord midline (mean 19.9 +/- 4.9 axons per 40-microm-thick section). Detailed single-axon reconstructions revealed that most ipsilaterally projecting lateral CST axons terminated in ipsilateral gray matter. Notably, we found that the bouton-like swellings of many ipsilateral CST axons descending in the dorsolateral tract were located within Rexed's lamina IX, in close proximity to motoneuronal somata. Thus, bilateral projections of corticospinal axons originating from a single motor cortex could contribute to bilateral control of spinal motor neurons and to the highly evolved degree of fine motor control in primates. Furthermore, bilateral CST projections from a single motor cortex could represent a potential source of plasticity after injury, as well as a target of therapeutic effort in neural regeneration strategies.

Ultrastructural features of synapse from dorsal parvocellular reticular formation neurons to hypoglossal motoneurons of the rat

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 μm 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.

Inspiratory augmenting bulbospinal neurons express both glutamatergic and enkephalinergic phenotypes.

Many of the inspiratory augmenting (I-AUG) neurons of the rostral ventral respiratory group (rVRG) are premotor neurons that excite phrenic motor neurons during inspiration, probably by releasing glutamate. In the present study, we demonstrate that these neurons are indeed glutamatergic, in that their cell bodies contain vesicular glutamate transporter-2 (VGLUT2) mRNA and spinal terminals from neurons in the region of the rVRG contain VGLUT2 protein. We also demonstrate by using parallel in situ hybridization and immunocytochemical evidence that most rVRG inspiratory premotor neurons are enkephalinergic. After iontophoretic deposits of biotinylated dextran amine (BDA) in the area of the rVRG, many BDA-labeled terminals in the ventral horn of cervical spinal cord (C4-C5) were immunoreactive for enkephalin and VGLUT2. Injections of Fluoro-Gold amidst phrenic motor neurons in C4-C5 labeled neurons in the area of the rVRG that contained both VGLUT2 mRNA and preproenkephalin (PPE) mRNA as revealed by double in situ hybridization. Thirty-eight bulbospinal I-AUG neurons were recorded in the rVRG and filled with biotinamide by using the juxtacellular labeling technique. Every biotinamide-filled cell tested was positively labeled for VGLUT2 mRNA (n = 14), and most of the cells tested in a separate population exhibited PPE mRNA (16/18). We conclude that most of the phrenic inspiratory premotor neurons of the rVRG are glutamatergic neurons that may also release enkephalins.

Synaptic organization of GABAergic projections from the substantia nigra pars reticulata and the reticular thalamic nucleus to the parafascicular thalamic nucleus in the rat

The ventrolateral part of the parafascicular thalamic nucleus (PF), which is considered to take part in the control mechanism of orofacial motor functions, receives projection fibers not only from the dorsolateral part of the substantia nigra pars reticulata (SNr) but also from the ventral part of the reticular thalamic nucleus (RT) [Tsumori et al., Brain Res. 858 (2000) 429]. In order to better understand the influence of these fibers upon the PF projection neurons, the morphology, synaptology and chemical nature of them were examined in the present study. After ipsilateral injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) into the dorsolateral part of the SNr and biotinylated dextran amine (BDA) into the ventral part of the RT, overlapping distributions of PHA-L-labeled SNr fibers and BDA-labeled RT fibers were seen in the ventrolateral part of the PF. At the electron microscopic level, the SNr terminals made synapses predominantly with the medium to small dendrites and far less frequently with the somata and large dendrites, whereas approximately half of the RT terminals made synapses with the somata and large dendrites and the rest did with the medium to small dendrites of PF neurons. Some of single dendritic as well as single somatic profiles received convergent synaptic inputs from both sets of terminals. These terminals were packed with pleomorphic synaptic vesicles and formed symmetrical synapses. After combined injections of PHA-L into the dorsolateral part of the SNr, BDA into the ventral part of the RT and wheat germ agglutinin–horseradish peroxidase (WGA-HRP) into the ventrolateral part of the striatum or into the rostroventral part of the lateral agranular cortex, WGA-HRP-labeled neurons were embedded in the plexus of PHA-L- and BDA-labeled axon terminals within the ventrolateral part of the PF, where the PHA-L- and/or BDA-labeled terminals were in synaptic contact with single somatic and dendritic profiles of the WGA-HRP-labeled neurons. Furthermore, the SNr and RT axon terminals were revealed to be immunoreactive for γ-aminobutyric acid (GABA), by using the anterograde BDA tracing technique combined with immunohistochemistry for GABA. The present data suggest that GABAergic SNr and RT fibers may exert different inhibitory influences on the PF neurons for regulating the thalamic outflow from the PF to the cerebral cortex and/or striatum in the control of orofacial movements.

Premotor circuits controlling eyelid movements in conjunction with vertical saccades in the cat: I. The rostral interstitial nucleus of the medial longitudinal fasciculus.

Saccadic eye movements in the vertical plane are controlled by the rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF) and the interstitial nucleus of Cajal. Eye movements in the vertical direction are accompanied by concurrent upper eye lid movements. These gaze-related lid movements are produced by the levator palpebrae superioris muscle, whose motoneurons are located in the caudal central subdivision (CCS) of the oculomotor nucleus. The neural circuits that direct such gaze-related lid movements were examined by use of both conventional and dual neuronal tracing methods in the cat. Injections of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) into the area of the CCS revealed a distinctive subset of retrogradely labeled neurons located in the caudomedial portion of the riMLF. This subset of riMLF neurons was not labeled when injections were localized within the oculomotor nucleus proper, without involving the CCS. Injections of biotinylated dextran amine (BDA) that included this caudomedial riMLF region anterogradely labeled axons that projected profusely throughout the CCS. Labeled terminals were seen in close association with retrogradely labeled levator palpebrae motoneurons, which were primarily found contralateral to WGA-HRP muscle injections. Ultrastructural examination revealed that most BDA-labeled terminals contained clear spherical vesicles and formed asymmetrical synaptic contacts, primarily on the proximal dendrites of WGA-HRP-labeled motoneurons. A few had pleiomorphic vesicles. In summary, these results strongly suggest that the caudomedial part of the cat riMLF is a premotor center that monosynaptically controls lid movements in conjunction with vertical saccades.

Dopamine D2 receptors are present in prefrontal cortical afferents and their targets in patches of the rat caudate‐putamen nucleus

Glutamatergic neurons within the deep layers of the prefrontal cortex and dopaminergic neurons of the substantia nigra pars compacta preferentially terminate in patch-like regions within the caudate putamen nucleus (CPN). Activation of dopamine D2 receptors is known to potently modulate striatal glutamatergic transmission and may play a role in reward-based motor learning. To determine the cellular substrate for D2-mediated regulation of prefrontal corticostriatal transmission in striatal patches, we combined anterograde transport of biotinylated dextran amine (BDA) with immunogold-silver labeling of a D2 receptor antipeptide antiserum in rat brain. Injections centered in deep layers of the dorsal part of the anterior cingulate cortex, one of the prefrontal cortical regions, produced varicose axonal BDA labeling in a patch-like distribution in the dorsomedial CPN. Electron microscopy showed that in these patch compartments, BDA labeling was present exclusively in axons and terminals (total number = 581), 9% of which contained detectable D2-like immunoreactivity. Thirty percent of the BDA-labeled terminals formed asymmetric excitatory synapses with dendritic spine heads, and the remainder were without recognizable junctions. The recipient spines were unlabeled or contained immunogold-silver particles for D2 receptors. A few of the D2-labeled spines also received convergent, often nonsynaptic contact from D2-labeled terminals resembling dopaminergic afferents. In addition, the corticostriatal terminals often apposed spiny and nonspiny neuronal profiles that contained D2 labeling. These results suggest that dopamine D2 receptors are strategically positioned for presynaptic and postsynaptic modulation of prefrontal corticostriatal excitation of spiny neurons in striatal patches. The findings have direct implications for D2-mediated control of reward-related motor learning.

Light and electron microscopic observations of a direct projection from mesencephalic trigeminal nucleus neurons to hypoglossal motoneurons in the rat

A direct projection from rat mesencephalic trigeminal nucleus (Vme) neurons to the hypoglossal nucleus (XII) motoneurons was studied using a double labeling method of anterogradely biotinylated dextran amine (BDA) tracing combined with retrogradely horseradish peroxidase (HRP) transport at both light and electron microscopic levels. BDA was iontophoresed unilaterally into the caudal Vme, and 7 days later HRP was injected into the ipsilateral tongue to label hypoglossal motoneurons. The BDA-labeled fibers were seen descended along Probst’ tract and were traced to the caudal medulla. In this course, the fibers gave off axon collaterals bearing varicosities in the trigeminal motor nucleus (Vmo), the parvicellular reticular formation (PCRt), the dorsomedial portions of the subnuclei of oralis (Vodm) and interpolaris (Vidm) and in the XII ipsilaterally. The labeling of terminals was most dense in the PCRt at the levels of caudal pons and rostral medulla, which displayed a ‘dumbbell-shaped’ form in the transverse planes. In the XII, labeled terminals were distributed mainly in the dorsal compartment of the nucleus. One hundred sixty-eight appositions made by BDA-labeled terminals on HRP-labeled motoneurons were seen in the dorsal compartment (71%) and in the lateral subcompartment (24%) of the ventral XII. Under electron microscopy BDA-labeled boutons containing clear, spherical synaptic vesicles were found to form synaptic contacts with the somata and dendrites of hypoglossal motoneurons with asymmetric specializations. The present study provides new evidence that the trigeminal proprioceptive afferent neurons terminate in the XII and make synaptic contacts with their motoneurons.

Axonal projections and synapses from the supratrigeminal region to hypoglossal motoneurons in the rat

Neural circuits from the supratrigeminal region (Vsup) to the hypoglossal motor nucleus were studied in rats using anterograde and retrograde neuroanatomical tracing methodologies. Iontophoretic injection of 10% biotinylated dextran amine (BDA) unilaterally into the Vsup anterogradely labeled axons and axon terminals bilaterally in the hypoglossal nucleus (XII) as well as other regions of the brainstem. In the ipsilateral XII, the highest density of BDA labeling was found in the dorsal compartment and the ventromedial subcompartment of the ventral compartment, where BDA labeling formed a dense, patchy distribution. Microinjection of 20% horseradish peroxidase (HRP) ipsilaterally or bilaterally into the tongue resulted in retrograde labeling of XII motoneurons confined to the dorsal and ventral compartments of the hypoglossal motor nucleus. Under light microscopical examination, BDA-labeled terminals were observed closely apposing the somata and primary dendrites of HRP-labeled hypoglossal motoneurons. Two hundred and sixty-five of these BDA-labeled terminals were examined at the ultrastructural level. One hundred and twelve BDA-labeled axon terminals were observed synapsing with either the somata (39%, 44/112) or the large or medium-size dendrites (61%, 68/112) of retrogradely labeled hypoglossal motoneurons. Axon terminals containing spherical vesicles (S-type) formed asymmetric synapses with HRP-labeled hypoglossal motoneuron dendrites. In contrast to this, F F-type axon terminals, containing flattened vesicles, formed symmetric synapses with both the somata and dendrites of HRP-labeled hypoglossal motoneurons with a preponderance of the contacts on their somata. Axon terminals containing pleomorphic vesicles (F P-type) were noted forming both symmetric and asymmetric synapses with HRP-labeled hypoglossal motoneuron somata and dendrites. The present study provides anatomical evidence of neuronal projections and synaptic connections from the supratrigeminal region to hypoglossal motoneurons. These data suggest that the supratrigeminal region, as one of the premotor neuronal pools of the hypoglossal nucleus, may coordinate and modulate the activity of tongue muscles during oral motor behaviors.

A di-synaptic projection from the lateral cerebellar nucleus to the laterodorsal part of the striatum via the central lateral nucleus of the thalamus in the rat

We have examined a cerebello–thalamo–striatal pathway from the lateral cerebellar nucleus (LCN) to the laterodorsal part of the striatum (LDS) through the central lateral nucleus (CL) using light and electron microscopy through the employment of a combination of anterograde and retrograde tracing techniques. Biotinylated dextran amine (BDA) was injected into the unilateral LCN, and used as an anterograde tracer. Cholera toxin B subunit (CTb), used for light microscopy, and wheat germ agglutinin–horseradish peroxidase (WGA-HRP), used for electron microscopy, were injected into the contralateral LDS as retrograde tracers. Light microscopic analysis showed a good overlap of the distribution of BDA-labeled axon terminals and CTb-labeled neurons in the middle third of the CL in both dorsoventral and rostrocaudal axes on the LDS injection side. Electron microscopy confirmed the presence of direct synaptic contacts between BDA-labeled terminals and WGA-HRP-labeled dendrites in the CL.

Possible pathways through which neurons of the shell of the nucleus accumbens influence the outflow of the core of the nucleus accumbens.

The nucleus accumbens (Acb), a major sector of the ventral striatum, is considered to be an integral part of the striatal complex. The Acb has been shown to be composed of two subdivisions, core and shell, which are distinguishable in several aspects, suggesting that these two subdivisions play different functional roles. The aim of this study was to identify pathways of the efferents of the shell of the Acb to influence the outflow of the core of the Acb. Potential disynaptic projections of the shell to the core of the Acb were investigated in chloral hydrate-anesthetized male Sprague-Dawley rats. Following ipsilateral injections of biotinylated dextran amine (BDA) into the shell of the Acb and cholera toxin B subunit (CT-B) into the core, strong overlapping distributions of BDA-labeled terminals and CT-B-labeled neuronal cell somata were found in the medial part of the ventral tegmental area, medial part of the lateral hypothalamic area, and dorsolateral part of the basolateral amygdaloid nucleus. The significance of multiple sites of relay between the efferents of the shell and the afferents of the core of the Acb at different levels of the neuraxis may be related to the functional specificity of each relay site.

Mu-opioid receptors are present in vagal afferents and their dendritic targets in the medial nucleus tractus solitarius.

Ligands of the mu-opiate receptor (MOR) are known to influence many functions that involve vagal afferent input to the nucleus tractus solitarius (NTS), including cardiopulmonary responses, gastrointestinal activity, and cortical arousal. The current study sought to determine whether a cellular substrate exists for direct modulation of vagal afferents and/or their neuronal targets in the NTS by ligands of the MOR. Anterograde tracing of vagal afferents arising from the nodose ganglion was achieved with biotinylated dextran amine (BDA), and the MOR was detected by using antipeptide MOR antiserum. The medial subdivision of the intermediate NTS was examined by electron microscopy for the presence of peroxidase-labeled, BDA-containing vagal afferents and immunogold MOR labeling. MOR was present in both presynaptic axon terminals and at postsynaptic sites, primarily dendrites. In dendrites, MOR immunogold particles usually were located along extrasynaptic portions of the plasma membrane. Of 173 observed BDA-labeled vagal afferent axon terminals, 33% contained immunogold labeling for MOR within the axon terminal. Many of these BDA-labeled terminals formed asymmetric, excitatory-type synapses with dendrites, some of which contained MOR immunogold labeling. MORs were present in 19% of the dendrites contacted by BDA-labeled terminals but were present rarely in both the vagal afferent and its dendritic target. Together, these results suggest that MOR ligands modulate either the presynaptic release from or the postsynaptic responses to largely separate populations of vagal afferents in the intermediate NTS. These results provide a cellular substrate for direct actions of MOR ligands on primary visceral afferents and their second-order neuronal targets in NTS.

Presence of NMDA-type glutamate receptors in cingulate corticostriatal terminals and their postsynaptic targets.

The glutamatergic projection from the anterior cingulate cortex to the medial caudate-putamen nucleus (CPN) has been implicated in motor and cognitive functions, many of which are potently modulated by activation of N-methyl-D-aspartate subtype of glutamate receptors (NMDARs). To determine the functional sites for NMDAR activation within this circuitry, we combined anterograde transport of biotinylated dextran amine (BDA) from deep layers of the rat anterior cingulate cortex with immunogold labeling of NMDAR subunit, NMDAR1, in the dorsomedial CPN. BDA-containing axons were seen in patch-like clusters in a neuropil that showed more uniform immunogold-silver labeling for NMDAR1. Electron microscopy of these regions showed that BDA-labeling was present exclusively in axons and terminals, 23% (98 of 421) of which also contained NMDAR1-immunoreactivity (IR). BDA-labeled terminals often apposed NMDAR1-immunoreactive neuronal and glial profiles. These terminals also formed asymmetric excitatory-type synapses with dendritic spines. Of 155 anterogradely labeled axon terminals forming asymmetric synapses, 34% were with NMDAR1-labeled, and 66% with unlabeled dendritic spines. These results provide ultrastructural evidence for the involvement of NMDARs in presynaptic regulation of glutamate transmission, and in postsynaptic modulation of the excitability of spiny neurons in patch-like compartments of the dorsomedial CPN. These dual NMDAR-mediated actions are likely to play a major role in the acquisition of new behaviors and reward-related processes that have been associated with cortical input to the striatal patch compartments.