Retrograde Cell Body Labeling Research Articles

Dual projecting cells linking thalamic and cortical communication routes between the medial prefrontal cortex and hippocampus

The interactions between the medial prefrontal cortex (mPFC) and the hippocampus (HC) are critical for memory and decision making and have been specifically implicated in several neurological disorders including schizophrenia, epilepsy, frontotemporal dementia, and Alzheimer’s disease. The ventral midline thalamus (vmThal), and lateral entorhinal cortex and perirhinal cortex (LEC/PER) constitute major communication pathways that facilitate mPFC-HC interactions in memory. Although vmThal and LEC/PER circuits have been delineated separately we sought to determine whether these two regions share cell-specific inputs that could influence both routes simultaneously. To do this we used a dual fluorescent retrograde tracing approach using cholera toxin subunit-B (CTB-488 and CTB-594) with injections targeting vmThal and the LEC/PER in rats. Retrograde cell body labeling was examined in key regions of interest within the mPFC-HC system including: (1) mPFC, specifically anterior cingulate cortex (ACC), dorsal and ventral prelimbic cortex (dPL, vPL), and infralimbic cortex (IL); (2) medial and lateral septum (MS, LS); (3) subiculum (Sub) along the dorsal–ventral and proximal–distal axes; and (4) LEC and medial entorhinal cortex (MEC). Results showed that dual vmThal-LEC/PER-projecting cell populations are found in MS, vSub, and the shallow layers II/III of LEC and MEC. We did not find any dual projecting cells in mPFC or in the cornu ammonis (CA) subfields of the HC. Thus, mPFC and HC activity is sent to vmThal and LEC/PER via non-overlapping projection cell populations. Importantly, the dual projecting cell populations in MS, vSub, and EC are in a unique position to simultaneously influence both cortical and thalamic mPFC-HC pathways critical to memory. Significance statementThe interactions between mPFC and HC are critical for learning and memory, and dysfunction within this circuit is implicated in various neurodegenerative and psychiatric diseases. mPFC-HC interactions are mediated through multiple communication pathways including a thalamic hub through the vmThal and a cortical hub through lateral entorhinal cortex and perirhinal cortex. Our data highlight newly identified dual projecting cell populations in the septum, Sub, and EC of the rat brain. These dual projecting cells may have the ability to modify the information flow within the mPFC-HC circuit through synchronous activity, and thus offer new cell-specific circuit targets for basic and translational studies in memory.

Virus Injection to the Pituitary via Transsphenoidal Approach and the Innervation of Anterior and Posterior Pituitary of Rat.

The theory holds that the anterior pituitary in mammals receives humoral regulation. Previous studies have reported that the pars distalis of the anterior pituitary of several mammalian species contains substance P-, calcitonin gene-related peptide (CGRP)-, and galanin-like immunoreactive nerve fibers, but the origins of these nerve fibers are unclear. Removal of the pituitary gland, also called hypophysectomy, involves methods that access the pituitary gland via the transauricular or parapharyngeal pathways. However, these methods are not applicable for viral tracer injection to investigate the innervation of the anterior pituitary. The transauricular technique leads to inaccuracies in locating the pituitary gland, while the parapharyngeal approach causes high mortality in animals. Here, we introduce a protocol that accesses the pituitary gland in the rat via the transsphenoidal pathway. This method imitates surgical manipulations such as endotracheal intubation and sphenoid bone drilling, which involve the use of custom-made devices. Using the transsphenoidal pathway greatly improves the survival rate of rats because no additional dissection of blood vessels and nerves is required. Moreover, the pituitary gland can be viewed clearly and directly during the operation, making it possible to accurately inject pseudorabies virus (PRV) 152-expressing enhanced green fluorescent protein (EGFP) into the anterior or posterior pituitary, respectively. After injecting PRV 152 into the anterior pituitary, we found no evidence of direct innervation of the anterior pituitary in the rat brain. However, PRV 152 injection into the posterior pituitary revealed retrograde transneuronal cell bodies in many brain areas, including the CA1 field of the hippocampus, the basolateral amygdaloid nucleus, posterior part (BLP), the arcuate hypothalamic nucleus (Arc), the dorsal portion of the dorsomedial hypothalamic nucleus (DMD), the suprachiasmatic nucleus (SCh), and the subfornical organ (SFO). In the present study, we provide a description of a possible model of hypophysectomy or pituitary injection, and identify brain regions involved in regulating the rat pituitary gland using transneuronal retrograde cell body labeling with PRV.

Motor Neuron Regeneration through End-to-Side Repairs Is a Function of Donor Nerve Axotomy

Over the past decade, a growing body of literature has emerged supporting the use of end-to-side (terminolateral) neurorrhaphy for the treatment of selected peripheral nerve injuries. It remains unclear, however, whether injury to the donor nerve is necessary to achieve significant regeneration through such repairs. End-to-side repair was studied in a rodent model in which the terminal limb of a transected peroneal nerve was sutured to the lateral aspect of the tibial nerve. Twenty-eight Lewis rats were randomized to four groups of seven animals each corresponding to incrementally greater donor nerve injuries as follows: group 1, conventional end-to-side neurorrhaphy; group 2, end-to-side neurorrhaphy with proximal crush injury; group 3, end-to-side neurorrhaphy with neurotomy; and group 4, end-to-end repair of transected peroneal nerve (positive control). At 12 weeks, retrograde labeling of cell bodies of the ventral horn demonstrated significant differences between experimental groups, with mean counts in group 4 (1237 +/- 171) > group 3 (522 +/- 204) > group 2 (210 +/- 132) > or = group 1 (126 +/- 146). This association between nerve injury and motor neuron counts was closely mirrored in quantitative assessments of peripheral nerve regeneration and normalized wet muscle masses. These data support the hypothesis that donor nerve injury is a prerequisite for significant motor neuronal regeneration across end-to-side repairs. Motor neuron regeneration through end-to-side repairs is optimized by deliberate transection of donor nerve axons.

Aldosterone-sensitive neurons in the nucleus of the solitary tract: bidirectional connections with the central nucleus of the amygdala.

The HSD2 (11-beta-hydroxysteroid dehydrogenase type 2-expressing) neurons in the nucleus of the solitary tract (NTS) of the rat are aldosterone-sensitive and have been implicated in sodium appetite. The central nucleus of the amygdala (CeA) has been shown to modulate salt intake in response to aldosterone, so we investigated the connections between these two sites. A prior retrograde tracing study revealed only a minor projection from the HSD2 neurons directly to the CeA, but these experiments suggested that a more substantial projection may be relayed through the parabrachial nucleus. Small injections of cholera toxin beta subunit (CTb) into the external lateral parabrachial subnucleus (PBel) produced both retrograde cell body labeling in the HSD2 neurons and anterograde axonal labeling in the lateral subdivision of the CeA. Also, injections of either CTb or Phaseolus vulgaris leucoagglutinin into the medial subdivision of the CeA labeled a descending projection from the amygdala to the medial NTS. Axons from the medial CeA formed numerous varicosities and terminals enveloping the HSD2 neurons. Complementary CTb injections, centered in the HSD2 subregion of the NTS, retrogradely labeled neurons in the medial CeA. These bidirectional projections could form a functional circuit between the HSD2 neurons and the CeA. The HSD2 neurons may represent one of the functional inputs to the lateral CeA, and their activity may be modulated by a return projection from the medial CeA. This circuit could provide a neuroanatomical basis for the modulation of salt intake by the CeA.

Brainstem projections to midline and intralaminar thalamic nuclei of the rat.

The projections from the brainstem to the midline and intralaminar thalamic nuclei were examined in the rat. Stereotaxic injections of the retrograde tracer cholera toxin beta -subunit (CTb) were made in each of the intralaminar nuclei of the dorsal thalamus: the lateral parafascicular, medial parafascicular, central lateral, paracentral, oval paracentral, and central medial nuclei; in the midline thalamic nuclei-the paraventricular, intermediodorsal, mediodorsal, paratenial, rhomboid, reuniens, and submedius nuclei; and, in the anteroventral, parvicellular part of the ventral posterior, and caudal ventral medial nuclei. The retrograde cell body labeling pattern within the brainstem nuclei was then analyzed. Nearly every thalamic site received a projection from the deep mesencephalic reticular, pedunculopontine tegmental, dorsal raphe, median raphe, laterodorsal tegmental, and locus coeruleus nuclei. Most intralaminar thalamic sites were also innervated by unique combinations of medullary and pontine reticular formation nuclei such as the subnucleus reticularis dorsalis, gigantocellular, dorsal paragigantocellular, lateral, parvicellular, caudal pontine, ventral pontine, and oral pontine reticular nuclei; the dorsomedial tegmental, subpeduncular tegmental, and ventral tegmental areas; and, the central tegmental field. In addition, most intralaminar injections resulted in retrograde cell body labeling in the substantia nigra, nucleus Darkschewitsch, interstitial nucleus of Cajal, and cuneiform nucleus. Details concerning the pathways from the spinal trigeminal, nucleus tractus solitarius, raphe magnus, raphe pallidus, and the rostral and caudal linear raphe nuclei to subsets of midline and intralaminar thalamic sites are discussed in the text. The discussion focuses on brainstem-thalamic pathways that are likely involved in arousal, somatosensory, and visceral functions.

Parabrachial nucleus projections to midline and intralaminar thalamic nuclei of the rat.

The projections from the parabrachial nucleus to the midline and intralaminar thalamic nuclei were examined in the rat. Stereotaxic injections of the retrograde tracer cholera toxin-beta (CTb) were made in each of the intralaminar nuclei of the dorsal thalamus (the lateral parafascicular, medial parafascicular, oval paracentral, central lateral, paracentral, and central medial nuclei), as well as the midline thalamic nuclei (the paraventricular, intermediodorsal, mediodorsal, paratenial, rhomboid, reuniens, parvicellular part of the ventral posterior, and caudal ventral medial nuclei). The retrograde cell body labeling pattern within the parabrachial subnuclei was then analyzed. The paracentral thalamic nucleus received an input only from the internal lateral parabrachial subnucleus. However, this subnucleus also projected to all the other intralaminar thalamic nuclei, except for the central lateral thalamic nucleus, which received no parabrachial afferent inputs. The external lateral parabrachial subnucleus projected to the lateral parafascicular, reuniens, central medial, parvicellular part of the ventral posterior, and caudal ventromedial thalamic nuclei. Following CTb injections in the paraventricular thalamic nucleus, retrogradely labeled cells were found in the central lateral, dorsal lateral, and external lateral parabrachial subnuclei. The medial and ventral lateral parabrachial subnuclei projected to the oval paracentral, parafascicular, and rhomboid thalamic nuclei. Finally, the waist area of the parabrachial nucleus was densely labeled after CTb injections in the parvicellular part of the ventral posterior thalamic nucleus. Nociceptive, visceral, and gustatory signals may reach specific cortical and other forebrain sites via this parabrachial-thalamic pathway.

CNS innervation of vagal preganglionic neurons controlling peripheral airways: a transneuronal labeling study using pseudorabies virus

The CNS cell groups that project to vagal preganglionic neurons which innervate the most distal part of the airways were identified by the viral retrograde transneuronal labeling method. Pseudorabies virus (PRV) was injected into the lung parenchyma of C8 spinal rats and after 5 days survival, brain tissue sections from these animals were processed for immunohistochemical detection of PRV. Retrogradely labeled parasympathetic preganglionic cells (first-order neurons) were seen mainly in the ventral medulla oblongata: the compact portion of the nucleus ambiguus and the area ventral to it. Occasionally, a few labeled cells were seen within the rostral part of the dorsal vagal nucleus. This labeling pattern correlated well with the retrograde cell body labeling seen following cholera toxin β-subunit (CT-b) injections in the lung parenchyma. PRV transneuronally labeled neurons (second-order and/or presumed third-order neurons) were found throughout the CNS with the characteristic labeling in the brainstem. Labeled neurons were identified along and just beneath the ventral medullary surface, and in nearby areas: the parapyramidal, retrotrapezoid, gigantocellular and lateral paragigantocellular reticular nuclei, as well as the caudal raphe nuclei (raphe pallidus, obscurus, and magnus). Several nucleus tractus solitarius (nTS) regions contained labeled cells including the commissural, medial, and ventrolateral nTS subnuclei. The A5 cell group and a small number of locus coeruleus neurons were also labeled. PRV-infected neurons were present in the Kölliker–Fuse and Barrington's nuclei. In the mesencephalon, neurons within the ventral periventricular gray matter were labeled. Labeling was present in the dorsal, lateral and paraventricular hypothalamic nuclei, and within the amygdaloid complex. In summary, the parasympathetic preganglionic neurons that innervate the peripheral airways are controlled by networks of lower brainstem and suprapontine neurons that lie in the same regions known to be involved in central regulation of autonomic functions.

Brain-stem afferents upon retinal projecting isthmo-optic and ectopic neurons of the pigeon centrifugal visual system demonstrated by retrograde transneuronal transport of rhodamine beta-isothiocyanate.

Brain-stem afferents to the n. isthmo-opticus (NIO) and ectopic neurons (EN) of the centrifugal visual system (CVS) were determined in the pigeon using the retrograde transneuronal transport of the fluorescent dye Rhodamine beta-isothiocyanate (RITC) after its intraocular injection. In other experiments, either RITC was injected into various periocular tissues (controls) or the retrograde tracer Fluoro-gold (FG) was injected stereotaxically in the NIO. Following intraocular injections, the RITC retrograde labeling of cell bodies was observed contralaterally in the NIO and EN and transneuronally in layers 9/10 of the optic tectum, area ventralis-Tsai, zona peri-NIII, mesencephalic and pontine reticular formation (PRF), n. linearis caudalis-raphe, and bilaterally within a region referred to as zona peri-n.NVI (Zp-n.NVI) immediately underlying the abducens nerve nucleus. None of the above structures were labeled after RITC periocular injections. The FG labeling revealed that the tectal efferent neurons were mainly medium-sized, multipolar cells whose dendrites extended superficially to retino-recipient tectal layers 6 and 5. Quantitative measurements of the distribution of layers 9/10 RITC-labeled neurons indicated the highest densities to be localized within the ventral tectum corresponding to the representation of the dorsal retina and inferior visual field. We suggest that visual and nonvisual brain-stem afferents upon NIO and EN may play a role in the proposed mechanism of the avian CVS in attention, ground-feeding behavior, and modulation of retinal sensitivity.

Central connections of the motor and sensory vagal systems innervating the trachea

Cholera toxin β-subunit was used as both a transganglionic and retrograde cell body tracer to determine respectively the central sensory and motor systems innervating the trachea in three mammalian species, dog, ferret and rat. A basic pattern was found in all three animals. Sensory fibers terminated in three subnuclei of the nucleus tractus solitarius (NTS) with the densest concentration localized in a restricted part of the medial part of the rostral NTS. Weaker projections were identified in the ventrolateral NTS subnucleus and sparse labeling was seen in the commissural NTS subnucleus. No labeling was identified in the area postrema. The pattern of retrograde cell-body labeling was also similar in all three species. Two main sites were labeled: the rostralmost part of the dorsal vagal nucleus and the rostral nucleus ambiguus (NA). In the NA, cell labeling was found in mainly in the ventral (or external) portion of the nucleus, but some labeled neurons were consistently found in the compact NA as well. In addition, labeled neurons were also seen in the dorsomedial part of the C1–C2 ventral hom. In summary, the central sites of termination of the sensory fibers and cells of origin innervating the trachea were similar in all three species.

Oxytocinergic innervation to the upper thoracic sympathetic preganglionic neurons in the rat. A light and electron microscopical study using a combined retrograde transport and immunocytochemical technique.

A combination of retrograde cell body labeling and immunohistochemistry was employed to elucidate how oxytocinergic fibers make contact with sympathetic preganglionic neurons (SPNs) in the rat spinal cord from T1 to T4. SPNs were labeled retrogradely using cholera toxin subunit B (CTb) or horseradish peroxidase-conjugated CTb. Oxytocin-immunoreactive (ir) fibers were found in the intermediate zone, including the sympathetic preganglionic subnuclei. In the central autonomic nucleus and the intercalated nucleus, brown-stained oxytocin-ir varicosities or terminals were frequently observed to stud black-stained dendrites of SPNs. Electron microscopical observations showed that oxytocin-ir terminals form synapses with dendrites or soma of the sympathetic preganglionic neurons. The terminals contained numerous small clear round vesicles and a few large, cored vesicles. These results clearly show that a large proportion of SPNs are innervated by oxytocin-containing fibers. The origin of these fibers is discussed, and it is concluded that they are probably descending fibers from the paraventricular nucleus of the hypothalamus.

CNS innervation of airway-related parasympathetic preganglionic neurons: a transneuronal labeling study using pseudorabies virus

The CNS cell groups that innervate the tracheal parasympathetic preganglionic neurons were identified by the viral retrograde transneuronal labeling method. Pseudorabies virus (PRV) was injected into the tracheal wall of C8 spinal rats and after 4 days survival, brain tissue sections from these animals were processed for immunohistochemical detection of PRV. Retrogradely labeled parasympathetic preganglionic neurons were seen in three sites in the medulla: the compact portion of the nucleus ambiguus, the area ventral to the nucleus ambiguus, and the rostralmost portion of the medial nucleus tractus solitarius (NTS); this labeling pattern correlated well with the retrograde cell body labeling seen following cholera toxin ß-subunit injections in the tracheal wall. PRV transneuronally labeled neurons were found throughout the CNS with the most abundant labeling concentrated in the ventral medulla oblongata. Labeled neurons were identified along the ventral medullary surface, and in nearby areas including the parapyramidal, retrotrapezoid, gigantocellular and lateral paragigantocellular reticular nuclei as well as the caudal raphe nuclei (raphe pallidus, obscurus, and magnus). Serotonin (5-HT) neurons of the caudal raphe complex (B1–B3 cell groups) and ventromedial medulla were labeled as well as a few C1 adrenergic neurons. The A5 cell group was the major noradrenergic area labeled although a small number of locus coeruleus neurons were also labeled. Several NTS regions contained labeled cells including the commissural, intermediate, medial, central, ventral, and ventrolateral subnuclei. PRV infected neurons were present in the Kölliker-Fuse and Barrington's nuclei. In the rostral mesencephalon, the precommissural nucleus of the dorsal periventricular gray matter was labeled. Labeling was present in the dorsal, lateral and paraventricular hypothalamic nuclei. In summary, the airway parasympathetic preganglionic neurons are innervated predominantly by a network of lower brainstem neurons that lie in the same regions known to be involved in respiratory and cardiovascular regulation. These findings are discussed in relationship to some of the potential CNS mechanisms that may be operative in airway disorders as well as potentially involved in certain fatal respiratory conditions such as Ondine's curse and sudden infant death syndrome (SIDS).

Bronchomotor vagal preganglionic cell bodies in the dog: an anatomic and functional study.

A previous study in our laboratory demonstrated that the stimulation with microinjection of DL-homocysteic acid of cell bodies in the rostral portion of the external formation of the nucleus ambiguus (Aext) increased total lung resistance in dogs. In the present study anatomic experiments were conducted in dogs to determine if the rostral Aext contains vagal preganglionic cell bodies that give rise to axons in the pulmonary branches of the vagus nerve. The application of horseradish peroxidase (HRP) to either the pulmonary branches or the vagus at a point between the pulmonary branches and the cardiac branches resulted in retrograde labeling of cell bodies in both rostral Aext and the dorsal motor nucleus of the vagus (DMN). On the other hand, application of HRP to the vagus at a point below the pulmonary branches did not result in any retrogradely labeled cell bodies in rostral Aext but did result in labeled cell bodies in DMN. In another series of experiments DL-homocysteic acid (2.5 nmol in 25 nl) was microinjected at sites in rostral Aext and DMN. As we previously reported the injection of DL-homocysteic acid in rostral Aext increased total lung resistance. In contrast, in the same animals, the injection of DL-homocysteic acid in DMN did not change total lung resistance. We conclude that bronchomotor vagal preganglionic cell bodies are located in rostral Aext but not in DMN. The functional significance of vagal preganglionic cell bodies in DMN whose axons contribute to the pulmonary branches of the vagus nerve remains to be determined.

Transneuronal labeling of spinal interneurons and sympathetic preganglionic neurons after pseudorabies virus injections in the rat medial gastrocnemius muscle

The distribution of retrogradely and transneuronally labeled neurons was studied in CNS of rats 4 days after injections of the Bartha strain of pseudorabies virus (PRV) into the medial gastrocnemius (MG) muscle. Tissue sections were processed for immunohistochemical detection of PRV. Retrogradely labeled cells were identified in the ipsilateral MG motor colum in the caudal L4 and the L5 spinal segments. In order to evaluate the efficacy of PRV retrograde cell body labeling, the number of PRV retrogradely labeled neurons in the MG motor column was compared to the number labeled with two conventional retrograde cell body markers — Fluoro-Gold and cholera toxin-HRP. A ratio of 1:3 representing medium-sized (< 30 μm) versus large neurons (>30 μm) was found in the Fluoro-Gold dye experiments; a 1:2 ratio was seen in the PRV experiments. In contrast, when cholera toxin-HRP was used as a retrograde marker, mainly large neurons were labeled; the medium-to-large cell body ratio was 1:10 suggesting cholera toxin-HRP may have a greater affinity for the terminals of α-motoneurons as opposed to γ-motoneurons. Transneuronally labeled cells were identified in the L1–L6 spinal gray matter, intermediolateral cell column (T11-L2), lateral spinal nucleus and medial part of lamina VII in C4 and C5 spinal segments, brainstem (caudal raphe nuclei, rostral ventrolateral medulla, A5 cell group, paralemniscal nucleus, locus coeruleus, subcoeruleus nucleus, red nucleus) and paraventricular hypothalamic nucleus. In the L5 spinal cord, transneuronally labeled neurons were seen in the ipsilateral spinal laminae I and II and bilaterally in spinal laminae IV–VIII, and X. Similar results were obtained in rats that had chronic unilateral L3–L6 dorsal rhizotomies indicating most of the labeling was due to retrograde transneuronal cell body labeling. In order to determine whether PRV was transported into the spinal cord by the dorsal root axons, the ipsilateral dorsal root ganglia (DRGs) were examined for PRV immunoreactivity; none was found. However, using the polymerase chain reaction, viral DNA was shown to be present in the ipsilateral DRGs indicating that some of spinal cord cell body labeling may have resulted from anterograde transneuronal labeling, as well.

Internuclear neurons in the ocular motor system of frogs

Medial and lateral rectus motoneurons of frogs were localized after retrograde labeling with horseradish peroxidase (HRP) injected in the medial rectus muscle or applied on the cut end of the abducens nerve. Coordinates of these cell columns were used as target areas for the injection of small amounts of HRP (20-60 nl) and [3H]leucine (25-40 nl) and as search areas for retrogradely and anterogradely labeled internuclear neurons (INT) in in vivo and in vitro experiments. HRP injection in the medial rectus subdivision of the oculomotor nucleus (n = 6) resulted in retrograde labeling of cell bodies in the contralateral principal abducens nucleus. On the average about 16 cells per animal were found. Somatic diameters were about 13.5 +/- 2.8 microns (n = 32). The number and the size of these abducens internuclear neurons (AbINT) are smaller than those of lateral rectus motoneurons (n = 75; diameter: 19 +/- 3.2 microns). A crossed projection of AbINT to medial rectus motoneurons in the contralateral oculomotor nucleus is further supported by autoradiographic results. Following injection of [3H]leucine into the abducens nucleus, a high density of silver grains was visible within the contralateral oculomotor nucleus, mainly in the caudal part of the oculomotor nucleus, where medial rectus motoneurons are located. Injection of [3H]leucine in vivo (n = 4) and in vitro (n = 3) resulted in a similar high density of silver grains within the contralateral oculomotor nucleus, but the background level of silver grains was significantly higher after in vitro (264 +/- 38/2,500 microns2) than after in vivo injections (195 +/- 17/2,500 microns2). HRP injection in the principal abducens nucleus (n = 9) resulted in retrograde labeling of cell bodies in the medial rectus subdivisions of the bilateral oculomotor nuclei. Ipsilateral projections predominated, with about 10 (+/- 8) labeled cells over contralateral projections (about 3 +/- 2). Average diameters of these oculomotor internuclear neurons (OcINT) were again smaller (10.8 +/- 2 microns; n = 18) than those of medial rectus motoneurons (14.4 +/- 3 microns; n = 52). In addition, retrogradely labeled cells were consistently encountered in the bilateral vestibular nuclei, the cerebellar nuclei, the dorsal brainstem caudal to the abducens nuclei, and ipsilaterally in the pretectum. Most of the vestibular neurons were located in the rostral part of the vestibular nuclear complex. These neurons might constitute part of the three-neuronal arc of the vestibulo-ocular reflex in the frog. Labeled cells in the pretectum were restricted to the ipsilateral posterior thalamic nucleus (P).(ABSTRACT TRUNCATED AT 400 WORDS)

Representation of the cecum in the lateral dorsal motor nucleus of the vagus nerve and commissural subnucleus of the nucleus tractus solitarii in rat

Motor fibers of the accessory celiac and celiac vagal branches are derived from the lateral columns of the dorsal motor nucleus of the vagus nerve. These branches also contain sensory fibers that terminate within the nucleus of the tractus solitarii. This study traces the innervation of the intestines by using the tracer cholera toxin-horseradish peroxidase. In 53 rats, the tracer was injected into either the stomach, duodenum, jejunum, terminal ileum, cecum, or ascending colon. With all cecal injections, prominent retrograde labeling of cell bodies occurred bilaterally in the lateral columns of the dorsal motor nucleus of the vagus nerve above, at, and below the level of the area postrema. Dendrites of laterally positioned neurons projected medially and rostrocaudally within the dorsal motor nucleus of the vagus nerve and dorsomedially into both the medial subnucleus and parts of the commissural subnucleus of the nucleus of the tractus solitarii. Sensory terminal labeling occurred in the dorsolateral commissural subnucleus at the level of the rostral area postrema and the medial commissural subnucleus caudal to the area postrema. Additionally, there was sensory terminal labeling within a small confined area of the dorsomedial zone of the nucleus of the tractus solitarii immediately adjacent to the fourth ventricle at a level just anterior to the area postrema. Stomach injections labeled motoneurons of the medial column of the entire rostrocaudal extent of the dorsal motor nucleus of the vagus nerve and a sensory terminal field primarily in the subnucleus gelatinosus, with less intense labeling extending caudally into the medial and ventral commissural subnuclei. Dendrites of gastric motoneurons project rostrocaudally and mediolaterally within the dorsal motor nucleus of the vagus nerve and dorsolaterally within the nucleus of the tractus solitarii. They are most pronounced at the level of the rostral area postrema where many dendrites course dorsolaterally terminating primarily within the subnucleus gelatinosus. Injections of the duodenum labeled a small number of the cells within the medial aspects of the dorsal motor nucleus of the vagus nerve. Jejunal, ileal, and ascending colon injections labeled cells sparsely within the lateral aspects of the dorsal motor nucleus of the vagus nerve bilaterally. No afferent terminal labeling was evident after injection of these areas of the bowel.(ABSTRACT TRUNCATED AT 400 WORDS)

CNS projections to the pterygopalatine parasympathetic preganglionic neurons in the rat: a retrograde transneuronal viral cell body labeling study

The retrograde transneuronal viral cell body labeling method was used to study the CNS nuclei that innervate the parasympathetic preganglionic neurons which project to the pterygopalatine ganglion. Small injections of a suspension of pseudorabies virus (PRV) were made in the pterygopalatine ganglion of rats and after 4 days their brains were processed for immunohistochemical detection of PRV. Some of the tissues were stained with a dual immunofluorescence method that permitted the visualization of PRV and neurotransmitter enzyme or serotonin immunoreactivity in the same cell. Retrograde cell body labeling was detected in the ipsilateral ventrolateral medulla oblongata in the region that has been termed the superior salivatory nucleus. This area was the same region that was retrogradely labeled after Fluoro-Gold dye injections in the pterygopalatine ganglion. Retrograde transneuronally infected cell bodies that provide putative afferent inputs to the pytergopalatine parasympathetic preganglionic neurons were mapped throughout the brain. In the medulla oblongata, transneuronally labeled neurons were seen in the nucleus tractus solitarii, dorsomedial part of the spinal trigeminal nucleus and gigantocellular reticular nucleus. In most experiments, some A1 catecholamine cells and serotonin neurons of the raphe magnus, raphe pallidus, raphe obscurus, and parapyramidal nuclei were labeled. In the pons, labeled cells were found in the parabrachial nucleus, A5 catecholamine cell group, and non-catecholamine part of the subcoeruleus region. In the midbrain, cell body labeling was located in the central gray matter and retrorubral field. In the diencephalon, labeling was found mainly in the hypothalamus. The areas included the lateral hypothalamic area, lateral preoptic area, dorsomedial and paraventricular hypothalamic nuclei, and ventral zona incerta. Contralateral second order cell body labeling was seen in the tuberomammillary nucleus of the hypothalamus. Some of these cells were histidine decar☐ylase-immunoreactive. In the forebrain, the bed nucleus of the stria terminalis, substantia innominata, and an area of the cerebral cortex called the amygdalopiriform transition zone were labeled.

Afferent fibers in the hypoglossal nerve: A horseradish peroxidase study in the cat

The existence of afferent fibers in the cat hypoglossal nerve was studied by transganglionic transport of horseradish peroxidase (HRP). Injections of wheat germ agglutinin-conjugated HRP (WGA-HRP) into the hypoglossal nerve resulted in some retrograde labeling of cell bodies within the superior ganglia of the ipsilateral glossopharyngeal and vagal nerves. A few labeled cell bodies were also present ipsilaterally within the inferior ganglion of the vagal nerve and the spinal ganglion of the C1 segment. Some of the labeled glossopharyngeal and vagal fibers reached the nucleus of the solitary tract by crossing the dorsal portion of the spinal trigeminal tract. Others distributed to the spinal trigeminal nucleus pars interpolaris and to the ventrolateral part of the medial cuneate nucleaus by descending through the dorsal portion of the spinal trigeminal tract. In the spinal cord these descending fibers, intermingling with labeled dorsal root fibers, distributed to laminae, I, IV–V and VII–VIII of the C1 and C2 segments. Additional HRP experiments revealed that the fibers in laminae VII–VIII originate mainly from dorsal root of the C1 segment.

Innervation of the spleen in the rat: Evidence for absence of afferent innervation

Catecholaminergic fibers in the spleen have been well characterized in the rat and this innervation is believed to be an important source of modulation of the immune system. The presence or role of afferent feedback from the spleen has not been systematically investigated. We have examined whether the spleen receives afferent innervation from sensory ganglia and also have assessed the sources of efferent innervation to the spleen in the rat. The fluorescent retrograde anatomical tracers fluoro-gold (FGo) or fast blue (FB) were injected into the spleens of adult female rats and dorsal root, sympathetic chain, nodose, and celiac-mesenteric plexus ganglia were collected. In additional animals, the spleen was either injected with the anatomical tracer wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or else regular HRP was applied to the cut end of the splenic nerve. Also, we examined the effects of cutting the splenic nerve on the retrograde labeling of cell bodies in the ganglia and on the catecholamine histochemistry of the spleen. The neuroanatomical results were based primarily upon the tracer FGo and verified that the celiac-mesenteric plexus ganglia provide a major efferent input to the spleen. Furthermore, lower thoracic sympathetic chain ganglia provide an additional and substantial efferent supply to the spleen. Cutting of the splenic nerve prevented retrograde labeling of cell bodies in the celiac-mesenteric plexus ganglia and sympathetic chain ganglia of rats injected with tracers into the spleen and also eliminated catecholamine histofluorescence in the spleen. In terms of afferent labeling, the results with FGo indicated that there were no cell bodies labeled in afferent ganglia following splenic injections. Similar injections of FGo into the hind foot pad produced numerous intensely labeled cells in the ipsilateral lower lumbar dorsal root ganglia. The only suggestion of positive labeling of any sensory cell bodies produced by FGo injections into the spleen was when the tracer was injected into the hilar region in close proximity to the splenic vessels. Injections of WGA-HRP into the spleen and exposing the cut end of the splenic nerve to HRP substantiated the observations based upon the tracer FGo. In contrast to the other tracers, FB produced faintly labeled cell bodies in the dorsal root ganglia, irrespective of their spinal levels, and this was not eliminated by cutting the splenic nerve. Thus, FB produced false positive labeling of afferent cell bodies following splenic injections. These results indicate that with the possible exception of the hilar region, the spleen receives virtually no afferent supply as determined by these sensitive methods but, rather, receives an extensive efferent innervation from both the celiac-mesenteric and sympathetic chain ganglia. The effects of nerve section indicate that virtually all of the efferent fibers to the spleen travel in the splenic nerve.

CNS cell groups regulating the sympathetic outflow to adrenal gland as revealed by transneuronal cell body labelling with pseudorabies virus

The CNS cell groups that innervate the sympathoadrenal preganglionic neurons of rats were identified by a transneuronal viral cell body labelling technique combined with neurotransmitter immunohistochemistry. Pseudorabies virus was injected into the adrenal gland. This resulted in retrograde viral infections of the ipsilateral sympathetic preganglionic neurons (T 4−T 13) and caused retrograde transneuronal cell body infections in 5 areas of the brain: the caudal raphe nuclei, ventromedial medulla, rostral ventrolateral medulla, A5 cell group, and paraventricular hypothalamic nucleus (PVH). In the spinal cord, the segmental distribution of virally infected neurons was the same as the retrograde cell body labeling observed following Fluoro-gold injections in the adrenal gland except there was almost a 300% increase in the number of cells labeled and a shift in cell group distribution. These results imply there are local interneurons that regulate the sympathoadrenal preganglionic neurons. In the medulla oblongata, serotonin (5-HT)-, substance P (SP)-, thyrotropin-releasing hormone-, Met-enkephalin-, and somatostatin-immunoreactive neurons of the raphe pallidus and raphe obscurus nuclei and the ventromedial medulla were infected. In the ventromedial and rostral ventrolateral medulla, immunoreactivephenylethanolamine- N-methyltransferase, SP, neuropeptide Y, somatostatin, and enkephalin neurons were infected. The A5 noradrenergic cells were labeled, as were some somatostatin-immunoreactive neurons in this area. In the hypothalamus, tyrosine hydroxylase- and SP-immunoreactive neurons of the dorsal parvocellular PVH were infected. Only a few immunoreactive vasopressin, oxytocin, Met-enkephalin, neurotensin, and somatostatin PVH neurons were labeled.

Further studies on the use of the fluorescent tracers fast blue and diamidino yellow: Effective uptake area and cellular storage sites

Some basic methodological issues concerning the use of the fluorescent tracers Fast blue (FB) and Diamidino yellow (DY) were studied using the projections of the red nucleus to the nucleus interpositus anterior (NIA) of the cerebellum of the cat. On standard Nissl-stained sections, it was possible to delineate 4 distinct zones at the FB and DY injection sites. Correlative studies of injection sites in the NIA and retrograde labeling of cell bodies in the contralateral red nucleus showed that effective uptake occurred only from the zone mechanically damaged by the injection needle (termed zone 0). The tracer remains in this zone during the post-injection survival. The limited uptake area for both tracers is a valuable feature for studies of restricted neuronal projections. However, the tracers are not suitable for use in quantitative studies, especially those concerning axonal collateralization. Perfusion with watersoluble fixatives did not alter the cellular storage site. In double-labeling experiments using horseradish peroxidase and DY, the HRP histochemistry induced an important “washing out” of DY and consequently, an underestimation of the number of labeled neurons.