Organization of the disynaptic pathway from the anteroventral cochlear nucleus to the lateral superior olivary nucleus in the ferret

The medial nucleus of the trapezoid body (MNTB) is one of three major nuclei of the superior olivary complex and provides an important inhibitory input from the contralateral ear to the lateral superior olivary nucleus (LSO) in the initial binaural pathway for coding interaural intensity differences. The major input to the MNTB from the contralateral anteroventral cochlear nucleus (AVCN) involves giant, calyx-like endings that have a one-to-one relationship with cells in the MNTB as confirmed in the ferret in this study. The main objective of the present study was to define the subsequent organization of projections from cells receiving these calyx-like endings. Several anatomical tracers (Phaseolus vulgaris leucoagglutinin, dextran-biotin, and biocytin) were used that are transported both anterogradely and retrogradely within neuronal projections in order to define the organization of MNTB connections with the LSO in the adult ferret. Analysis focused on determining the topography in both the transverse and longitudinal planes of the projections. Focal tracer injections in the LSO resulted in retrograde labeling of a long, narrow column of cells in the MNTB. The orientation and location of labeled cells was dependent on the medial-lateral position of the injection site. In the rostral-caudal dimension of MNTB, there was no such topographic relation between the injection site and the position of labeled cells. Labeled cells in the MNTB were distributed more or less evenly in a longitudinal column regardless of whether the injection site was restricted to the rostral, middle or caudal part of the LSO. In keeping with this pattern, tracer injections in the MNTB resulted in bands of labeled axons that distributed endings throughout the rostral-caudal axis of the LSO. These bands or sheets varied in medial-lateral position relative to the location of the injection site, but lacked any such rostral-caudal gradient. Thus, overall the MNTB-LSO projections have a convergent-divergent pattern of organization. While MNTB cells receive singular calyx-like endings from the AVCN, LSO cells receive projections from a long column of cells in the MNTB. Implications for processing interaural intensity differences are discussed.     

The topographical organization of descending projections from the central nucleus of the inferior colliculus in guinea pig

We describe the descending projections from the central nucleus of the inferior colliculus (CNIC) in guinea pig. Focal injections of the tracer biocytin, made in physiologically defined frequency regions of the CNIC, labelled laminated axonal terminal fields in the ipsilateral dorsal nucleus of the lateral lemniscus, and bilaterally in the ventral nucleus of the trapezoid body and the dorsal cochlear nucleus. Labelling was also present in the rostral periolivary nucleus, but we could not distinguish a clear border between the terminal fields in this nucleus and those in the ventral nucleus of the trapezoid body. Labelling observed in the ventral nucleus of the lateral lemniscus, and to a lesser extent in the dorsal nucleus of the lateral lemniscus, was accompanied by retrogradely labelled somata and therefore we cannot conclude unequivocally that the CNIC projects to these lemniscal nuclei. Where the labelling was ordered topographically, its position varied as a function of the best frequency at the injection site. High-frequency regions in the CNIC project to the medial parts of the ventral nucleus of the trapezoid body and dorsal cochlear nucleus, while low-frequency regions in the CNIC project to the lateral parts of the ventral nucleus of the trapezoid body and dorsal cochlear nucleus. Additional axonal labelling with terminal boutons, but with no apparent topographical arrangement, was present in the ipsilateral horizontal cell group, sagulum, and also bilaterally in the superficial granule cell layer of the ventral cochlear nucleus and layer 2 of the dorsal cochlear nucleus. Our findings are consistent with the existence of tonotopically organised feedback projections from the CNIC to the brainstem nuclei that project to it.     

Sources of GABAergic input to the inferior colliculus of the rat

We have studied the GABAergic projections to the inferior colliculus (IC) of the rat by combining the retrograde transport of horseradish peroxidase (HRP) and immunohistochemistry for gamma-amino butyric acid (GABA). Medium-sized (0.06-0.14 microliter) HRP injections were made in the ventral part of the central nucleus (CNIC), in the dorsal part of the CNIC, in the dorsal cortex (DCIC), and in the external cortex (ECIC) of the IC. Single HRP-labeled and double (HRP-GABA)-labeled neurons were systematically counted in all brainstem auditory nuclei. Our results revealed that the IC receives GABAergic afferent connections from ipsi- and contralateral brainstem auditory nuclei. Most of the contralateral GABAergic input originates in the IC and the dorsal nucleus of the lateral lemniscus (DNLL). The dorsal region of the IC (DCIC and dorsal part of the CNIC) receives connections mostly from its homonimous contralateral region, and the ventral region from the contralateral DNLL. The commissural GABAergic projections originate in a morphologically heterogeneous neuronal population that includes small to medium-sized round and fusiform neurons as well as large and giant neurons. Quantitatively, the ipsilateral ventral nucleus of the lateral lemniscus is the most important source of GABAergic input to the CNIC. In the superior olivary complex, a smaller number of neurons, which lie mainly in the periolivary nuclei, display double labeling. In the contralateral cochlear nuclei, only a few of the retrogradely labeled neurons were GABA immunoreactive. These findings give us more information about the role of GABA in the auditory system, indicating that inhibitory inputs from different ipsi- and contralateral, mono- and binaural auditory brainstem centers converge in the IC.     

Ultrastructural and immunocytochemical characterization of neurons in the rat ventral cochlear nucleus projecting to the inferior colliculus

Medium to large-giant multipolar neurons in the rat ventral cochlear nucleus were retrograde labelled after injection of the tracer Wheat Germ Agglutinin conjugated to Horse Radish Peroxidase into the contralateral cochlear nucleus. Light microscopy immunocytochemistry showed that 42.45% of these retrograde labelled neurons, generally strongly labelled with the tracer, were markedly glycine immunopositive, and that 57.55%, usually weakly retrograde labelled neurons, were immunonegative or weakly positive for glycine. These commissural neurons were generally GABA negative and variably immunopositive for glutamate. About 1/3rd of the commissural neurons had variably developed a rough endoplasmic reticulum whilst axo-somatic boutons covered 20-40% of the cell body. These cells were recognized as multipolar neurons of type I. Most of them were weakly glycine positive or even negative and a few appeared glycinergic. A little less than the remaining 2/3rds of the whole commissural population in the postero-ventral cochlear nucleus presented a surface which was 65-85% covered with synaptic boutons, among which some also appeared labelled. These cells were recognized as multipolar neurons of type II. Many microtubules and neurofilaments were present, free ribosomes being more numerous around Nissl bodies with short cisternae. A few low retrograde labelled type II were weakly or non glycinergic. A small number of large to giant neurons type II, strongly retrograde labelled, appeared to be glycine positive, consistently GABA negative and variably glutamate positive. A very small proportion of retrograde labelled neurons appeared having the characteristics of globular bushy neurons. Their weak labelling, however, suggests that they project by collaterals or thin axons to the contralateral cochlear nucleus. Spherical bushy cells in the rat anteroventral cochlear nucleus lack the nuclear capping of rough endoplasmic reticulum observed in the cat, and none was labelled after injection into the contralateral cochlear nucleus. Globular and spherical neurons were variably glutamate positive but glycine and GABA negative. In conclusion, the present study suggests that commissural neurons include a small number of strongly labelled large to giant glycinergic and presumably inhibitory type II and, less frequently type I. A large group of less heavily labelled commissural neurons of type I and II contain low levels or no glycine, which is probably used for metabolic purposes rather than as a neurotransmitter. This suggests that these neurons are presumably excitatory.     

Secondary vestibulo-oculomotor projections in larval sea lamprey: anterior octavomotor nucleus

The ventral octavolateral area of lampreys contains three nuclei: the anterior, intermediate and posterior octavomotor nuclei, formed of large neurons that are contacted by thick primary vestibular fibres. We used horseradish peroxidase (HRP) or fluorescein-dextran-amine (FDA) labelling to study the projections of the anterior octavomotor nucleus (AON) in the larval sea lamprey, Petromyzon marinus. The tracers were injected either in the AON, the oculomotor nucleus or the rostralmost spinal cord. HRP injection in the AON labelled thick axons that coursed to the basal mesencephalic tegmentum, where most decussate and project to the oculomotor nucleus and the third Müller cell. Electron microscopy confirmed that AON axons contact with the contralateral third Müller cell and with oculomotor neurons. Some AON axons run in the mesencephalic tegmentum and the ventral diencephalon. An AON axon was observed to run close to the axon of the contralateral third Müller cell, establishing what appeared to be en passant contacts. HRP injection in the AON also revealed commissural fibres projecting to the contralateral octavolateral area. HRP or FDA injections in the oculomotor nucleus labelled both large and small neurons of the AON, mostly contralateral to the injection site, as well as of cells in the intermediate octavomotor nucleus, mainly ipsilateral. HRP injection in the AON or in the rostral spinal cord did not reveal any projections from the AON to the spinal cord. Our results indicate that the pattern of octavo-oculomotor connections in the lamprey is different from that observed in other vertebrates.     

Colocalization of substance P or enkephalin in serotonergic neuronal afferents to the hypoglossal nucleus in the rat

The serotonergic innervation of the hypoglossal nucleus originates from the caudal raphe nuclei. Non-serotonergic neurons in the caudal raphe nuclei also project to the hypoglossal nucleus. We employed a triple-fluorescence technique to determine whether the substance P- or the enkephalin-containing neurons in the caudal raphe nuclei that projected to the hypoglossal nucleus also contained serotonin. Rhodamine latex microspheres were injected into the hypoglossal nucleus, and then serotonin and peptide dual-immunofluorescence was performed to colocalize perikarya containing serotonin, substance P, and rhodamine microspheres; or perikarya containing serotonin, enkephalin, and rhodamine microspheres. Our results demonstrate that most substance P-containing neuronal afferents to the hypoglossal nucleus colocalize serotonin. In contrast, few enkephalin-containing neuronal afferents to the hypoglossal nucleus also contain serotonin. These data suggest that substance P projections to the hypoglossal nucleus are a subset of serotonergic projections and that limited overlap exists between the populations of enkephalinergic and serotonergic neuronal afferents to the hypoglossal nucleus. Either substance P- or enkephalin-containing somata account for a very small proportion of non-serotonergic caudal raphe projections to the hypoglossal nucleus. Finally, these data demonstrate the medial tegmental field origins of the substance P projections and the enkephalin projections to the hypoglossal nucleus.     

Cholinergic and non-cholinergic afferents of the caudolateral parabrachial nucleus: a role in the long-term enhancement of rapid eye movement sleep

A single microinjection of the cholinergic agonist carbachol into the feline caudolateral parabrachial nucleus produces an immediate increase in state-independent ipsilateral ponto-geniculooccipital waves, followed by a long-term rapid eye movement sleep enhancement lasting 7-10 days. Using retrogradely-transported fluorescent carbachol-conjugated nanospheres and choline acetyltransferase immunohistochemistry, afferent projections to this injection site for long-term rapid eye movement sleep enhancement were mapped and quantified. Six regions in the brain stem contained retrogradely-labelled cells: the raphe nuclei, locus coeruleus, laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, parabrachial nucleus, and the pontine reticular formation. The retrogradely-labelled (rhodamine+) cells in the pontine reticular formation and pedunculopontine tegmental nucleus contributed the predominant input to the parabrachial nucleus injection site (34.3 +/- 5.3% and 28.4 +/- 5.6%, respectively), compared to the laterodorsal tegmental nucleus (5.8 +/- 3.8%), parabrachial nucleus (13.5 +/- 3.1%), raphe nuclei (12.9 +/- 2.7%), and locus coeruleus (5.1 +/- 2.4%). By comparison with findings of afferent input to the induction site for short-latency rapid eye movement sleep in the anterodorsal pontine reticular formation, the parabrachial nucleus injection site is characterized by a similar proportion of afferents, except that the raphe nuclei were found to provide more than a two-fold greater input. Retrogradely-labelled neurons quantified in these nuclear regions consisted of 21.5% double-labelled (rhodamine+/choline acetyltransferase+) cholinergic and 78.5% noncholinergic (rhodamine+/choline acetyltransferase-) cells. The pedunculopontine tegmental nucleus contributed the predominant (51.7 +/- 8.2%) cholinergic input, compared to laterodorsal tegmental nucleus (20.7 +/- 10.2%), parabrachial nucleus (23.1 +/- 7.5%), and pontine reticular formation (4.4 +/- 2.1%). A comparative analysis of the total retrogradely-labelled cells within each nuclear region which were also double-labelled showed the highest proportion in the laterodorsal tegmental nucleus (76.2 +/- 7.5%) compared to pedunculopontine tegmental nucleus (39.4 +/- 3.6%), parabrachial nucleus (37.3 +/- 2.8%), and pontine reticular formation (3.2 +/- 2.1%). These data indicate that while pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus neurons exert a powerful cholinergic influence on the injection site for long-term rapid eye movement enhancement, a major component of the afferent circuitry is non-cholinergic. Since the non-cholinergic input includes contributions from the locus coeruleus and raphe nuclei, it is probable that the caudolateral parabrachial nucleus contains cholinergic and aminergic afferent systems that participate in the long-term enhancement of rapid eye movement sleep.     

Projections from the nucleus isthmi to the visual and auditory centres in the frog, Rana esculenta

The lectin Phaseolus vulgaris leucoagglutinin was injected into the nucleus isthmi (NI) in order to study its anterograde and retrograde projections in the frog. The following areas of termination could be discerned in the brainstem: (1) Each of the five subnuclei of the torus semicircularis (TOS) received fibres from the NI. The projection was the most extensive on the three main subnuclei which disclosed also retrogradely labelled neurones on the side of injections. The subependymal subnuclei contained the least number of labelled fibres. (2) Both hemispheres of the optic tectum (TO) were supplied by fibres from the NI. Labelled fibres were more numerous on the side of injections, and preterminal and terminal fibres covered columnar-like areas in layers 8 and 9. Several retrogradely labelled neurones were found in layer 6. Relatively few labelled fibres were seen on the contralateral side. They formed patch-like areas of termination in layer 9. (3) The anterodorsal (AD) and anteroventral (AV) nuclei were reciprocally inter-connected with the NI. The fibre connections were less extensive on the contralateral side. In the rhombencephalon (4) the cochlear nucleus (CN) and (5) the superior olive (SO) were also reciprocally connected with the NI on both sides, but with much weaker projection on the side contralateral to injections. (6) Only a weak anterograde labelling was observed in the contralateral NI and in the ipsilateral reticular formation.     

Evidence for a GABAergic projection from the central nucleus of the amygdala to the brainstem of the macaque monkey: a combined retrograde tracing and in situ hybridization study

The central nucleus of the amygdala is interconnected with a variety of visceral and autonomic nuclei of the brainstem. These include the parabrachial nucleus, the nucleus of the solitary tract, the nucleus ambiguus and the dorsal motor nucleus of the vagus. Despite repeated attempts, neurochemical characterization of the major subcortical connections of the central nucleus has not yet been accomplished. Based on earlier immunohistochemical and in situ hybridization evidence indicating the presence of numerous GABAergic neurons in the macaque monkey central nucleus, we predicted that a sizeable portion of the descending projections may be GABAergic. We tested this hypothesis using a novel double labelling method with gold conjugated WGA-apoHRP as a retrograde tracer and in situ hybridization for detecting the mRNA that encodes the enzyme glutamic acid decarboxylase (GAD67) as a marker for GABAergic cells. Following WGA-apoHRP-gold injections into the brainstem, a large number of retrogradely labelled cells was observed in the medial and lateral divisions of the central nucleus. Of the retrogradely labelled cells observed in the medial division of the central nucleus, approximately half were double-labelled for GAD67 mRNA; about 30% double labelling was observed in the lateral division. These data support the view that a sizeable component of the central nucleus projection to the brainstem is GABAergic.     

Contralateral cortical projection to the mediodorsal thalamic nucleus: origin and synaptic organization in the rat

The origin of the corticothalamic projections to the contralateral mediodorsal nucleus, the collateralization of cortical fibers and their synaptic organization in the ipsi- and contralateral mediodorsal nuclei were investigated in adult rats with double retrograde fluorescent and anterograde tracing. After tracer injections in the mediodorsal nuclei on either side, neurons were retrogradely labeled in all the areas of the contralateral prefrontal cortex in which ipsilateral labeling was also observed. Contralateral corticothalamic cells accounted for 15% of the labeled neurons in the orbital and agranular insular areas, while their proportion was lower (3%) in the anterior cingulate cortex. Up to 70% of the contralateral cortical neurons were double labeled by bilateral injections in the mediodorsal nuclei. At the electron microscopic level, unilateral injections of biotinylated dextran-amine in the orbitofrontal cortex resulted in anterograde labeling of small terminals and a few large boutons in the ipsilateral mediodorsal nucleus, while only small boutons were identified contralaterally. The diameter of postsynaptic dendritic profiles contacted by labeled small cortical endings was significantly larger in the ipsilateral mediodorsal nucleus than contralaterally. These findings demonstrate that dense contralateral cortical projections to the mediodorsal nucleus derive from the orbital and agranular insular areas, and that crossed corticothalamic afferents are mostly formed by collaterals of the ipsilateral connections. Our observations also point out the heterogeneity of corticothalamic boutons in the rat mediodorsal nucleus and morphological differences in the synaptic organization of prefrontal fibers innervating the two sides, indicating that ipsilateral cortical afferents may be more proximally distributed than crossed cortical fibers on dendrites of mediodorsal neurons.     

Vagal innervation of the gastrointestinal tract arises from dorsal motor nucleus while that of the heart largely from nucleus ambiguus in the cat

The origin of medullary cells that form the cardiac vagal branch and the vagal branches in the lower thorax innervating the gastrointestinal (GI) tract was studied using horseradish peroxidase (HRP), a retrograde transport tracer in the cat. The distributions of parasympathetic postganglionic neurons of the heart were studied with acetylcholinesterase histochemistry. Intracardiac ganglionic neurons were found mainly in the connective tissue surrounding the base of the pulmonary arteries and in an area in and dorsal to the interatrial septum. Following injection of HRP into the subepicardum where most of the cardiac postganglionic neurons reside, 91% of the labelled neurons were found bilaterally distributed in the nucleus ambiguus (NA). A small population of labelled neurons was found in the dorsal motor nucleus of the vagus (DMV) and an intermediate zone (IZ) between the two nuclei. When HRP was injected into the left or right cardiopulmonary vagus branch, labelled neurons were found exclusively in the ipsilateral NA, DMV and IZ with a predominance in the NA. In the thorax, after they course around the heart, the left and right thoracic vagus nerves divides into a left and a right branch, respectively. The left branch of the left thoracic vagus joins the left branch of the right thoracic vagus to form the anterior vagus nerve at 3 cm above the diaphragm. The right branch of the right thoracic vagus nerve joins the right branch of the left thoracic vagus to form the posterior vagus nerve. After application of HRP into the right or the left branch of the left thoracic vagus, HRP labelled cells were found in the left DMV. Similarly, after application of HRP into the left or the right branch of the right thoracic vagus, labelled cells were found in the right DMV. On the other hand, when HRP was injected into the anterior vagus, labelled neurons were found bilaterally in the DMV. This suggests that all rostral branches of the thoracic vagus have their origin in the ipsilateral DMV, and intermixing occurs only at the caudal level near the diaphragm. Findings of the present experiments suggest that parasympathetic preganglionic neurons innervating the GI tract are located exclusively in the DMV while those of the heart are located mainly in the NA. Within the DMV, GI vagal neurons were found medially from the level 0-2.5 mm rostral to the obex. In contrast, cardiac vagal neurons were found in the lateral edge of the DMV at the level 0-1 mm rostral to the obex.     

Ultrastructural, physiological, and molecular defects in the inner ear of a gene-knockout mouse model for autosomal Alport syndrome

It is well documented that damage to the chick cochlea caused by acoustic overstimulation or ototoxic drugs is reversible. Second-order auditory neurons in nucleus magnocellularis (NM) are sensitive to changes in input from the cochlea. However, few experiments studying changes in NM during cochlear hair cell loss and regeneration have been reported. Chicks were given a single systemic dose of gentamicin, which results in maximal hair cell loss in the base of the cochlea after 5 days. Many new hair cells are present by 9 days. These new hair cells are mature but not completely recovered in organization by 70 days. We counted neurons in Nissl-stained sections of the brainstem within specific tonotopic regions of NM, comparing absolute cell number between gentamicin- and saline-treated animals at both short and long survival times. Our data suggest that neuronal number in rostral NM parallels hair cell number in the base of the cochlea. That is, after a single dose of gentamicin, we see a loss of both cochlear hair cells and NM neurons early, followed by a recovery of both cochlear hair cells and NM neurons later. These results suggest that neurons, like cochlear hair cells, can recover following gentamicin-induced damage.     

New growth of axons in the cochlear nucleus of adult chinchillas after acoustic trauma

This study determined the effect of acoustic overstimulation of the adult cochlea on axons in the cochlear nucleus. Chinchillas were exposed to an octave-band noise centered at 4 kHz at 108 dB sound pressure level for 1.75 h. One chinchilla was never exposed to the noise, and several others had one ear protected by an ear plug or prior removal of the malleus and incus. Exposure of unprotected ears caused loss of inner and outer hair cells and myelinated nerve fibers, mostly in the basal half of the cochlea. Cochlear nerve fiber degeneration, ipsilateral to the exposed ears, was traced to regions of the cochlear nucleus representing the damaged parts of the cochlea. In silver impregnations of a deafferented zone in the posteroventral cochlear nucleus, the concentration of axons decreased by 43% after 1 month and by 54% after 2 months. However, by 8 months, the concentration of thinner axons, with diameters of less than 0.46 microm, increased by 46-90% over that at 2 months. The concentration of axons with larger diameters did not change. Between 2 and 8 months small axonal endings appeared next to neuronal cell bodies. This later increase of thinner axons and endings is consistent with a reactive growth of new axons of relatively small diameter. The emergence of small perisomatic boutons suggests that the new axons formed synaptic endings, which might contribute to an abnormal reorganization of the central auditory system and to the pathological changes that accompany acoustic overstimulation.     

Neuronal degeneration of primary cochlear and vestibular innervations after local injection of sisomicin in the guinea pig

This paper reports on a dynamic study of the morphological changes within the cochlear and vestibular ganglia of the guinea pig after local application of Sisomicin in the inner ear. The treatment leads to a rapid, complete and irreversible destruction of the sensory cells in the cochlear and vestibular neuroepithelia. A progressive degeneration of the type I and type II afferent neurons, presenting a decreasing gradient from the base towards the apex of the cochlea, is rapidly observed and becomes almost complete as early as 15 days after the peripheral injury. Five months after the treatment the spiral ganglion cells have almost completely disappeared. At this time the vestibular ganglion cell density appears normal but the neurons exhibit important signs of alteration. Such damage to the cochlear and vestibular afferent neurons may result from either retrograde neuronal degeneration and/or direct neurotoxic effect of the drug. Thus the combination of the two mechanisms could lead to neuronal losses in spiral and Scarpa's ganglia after the local aminoglycoside intoxication of the inner ear. The difference in the time course of degeneration for these two afferent ganglia could be due to their specific susceptibilities or to their different anatomical locations.     

Localisation of ionotropic glutamate receptor subunits, and gamma-aminobutyric acid (GABA) in the monkey amygdala--a double immunolabelling and electron microscopic study

The distribution of ionotropic glutamate receptor subunits GluR1 and NMDAR1, and the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) was studied by immunocytochemistry, in the monkey amygdala. The basal and lateral nuclei contained a higher density of GluR1-positive neurons than the corticomedial and central groups of nuclei, and the accessory basal nucleus. A higher density of NMDAR1 immunopositive cell bodies was also present in the lateral nucleus, compared to the other nuclei of the amygdala. Large multipolar or fusiform projection neurons, and not small local circuit neurons were GluR1 or NMDAR1-positive, and less than 0.5% of the GluR1-positive cells were double labelled for GABA. In contrast, almost all GluR1-positive neurons were also labelled for NMDAR1 in double labelled sections and vice versa. Electron microscopy also showed that GluR1- and NMDAR1-positive cells had distinctive ultrastructural features, compared with GABAergic cells, ant that there was very rare colocalisation, between GABA, and GluR1 or NMDAR1-positive cell bodies or dendrites. It is likely that not all projection neurons were GluR1 or NMDAR1-positive, however, since GluR1 or NMDAR1-positive neurons were only 2-3 times as common as GABAergic cells, whereas it has been estimated that projection neurons outnumber GABAergic local circuit neurons by 4 to 1 (McDONALD and AUGUSTINE, 1993; PITKANEN and AMARAL, 1994).     

Breed differences in deafferentation-induced neuronal cell death and shrinkage in chick cochlear nucleus

Removal of functional presynaptic input can result in a variety of changes in postsynaptic neurons in the central nervous system, including altered metabolism, changes in neuronal cell size, and even death of the postsynaptic cell. Age-dependent neuronal cell death and shrinkage has been documented in second order auditory neurons in the chick brainstem (nucleus magnocellularis, NM) following cochlea removal (Born and Rubel, 1985. J. Comp. Neurol. 231, 435-445). Here we examined whether the extent of neuronal cell death and shrinkage is also breed-dependent. We performed unilateral cochlea removal on both hatchling and adult birds of either a broiler breed (Arbor Acres Cross) or egg layer breed (Hy-Line, H and N) and killed birds one week later. Changes in neuronal cell number and cross sectional area were determined from Nissl-stained sections. We observed 25% neuronal cell loss and a 15-20% decrease in neuronal cross sectional area after cochlea removal in either broiler or egg layer hatchling birds. In adult birds, however, neuronal cell loss is breed-dependent. Adult egg layer birds lose an average of 37% of NM neurons after cochlea removal, while adult broiler birds show no cell loss. In both breeds of adult birds, cochlea removal results in a 20% decrease in neuronal cross sectional area. These results suggest that analysis of differences between breeds as well as ages of birds will prove fruitful in determining how afferent input controls neuronal survival and metabolism.     

Projections of the dorsomedial nucleus of the intercollicular complex (DM) in relation to respiratory-vocal nuclei in the brainstem of pigeon (Columba livia) and zebra finch (Taeniopygia guttata)

Injections of neuronal tracers were made into the dorsomedial nucleus of the intercollicular complex (DM) of pigeons and zebra finches in order to investigate the projections of this nucleus which has long been implicated in respiratory-vocal control. Despite the fact that pigeons are nonsongbirds and zebra finches are songbirds, the projections were very similar in both species. Most descended throughout the brainstem, taking ventral and dorsal trajectories, which merged in the medulla. Those descending ventrally terminated upon the ventrolateral parabrachial nucleus (PBvl), the nucleus infraolivaris superior, a nucleus of the rostral ventrolateral medulla (RVL), and the nucleus retroambigualis (RAm). Those taking a dorsal trajectory via the occipitomesencephalic tract terminated in the tracheosyringeal part of the hypoglossal nucleus (XIIts), the suprahypoglossal region, and nucleus retroambigualis. There were also substantial projections throughout an arc extending between XIIts and RVL rostrally, and XIIts and RAm caudally. Neurons throughout this arc, which include inspiratory premotor neurons at levels straddling the obex and expiratory premotor neurons more caudally (in RAm), were retrogradely labeled from spinal injections. The DM projections were predominantly ipsilateral, but there were distinct contralateral projections to all the homologous nuclei in both species. All but the projections to PBvl and XIIts were reciprocal. In summary, the projections of DM suggest that it is able to influence all the key motor and premotor nuclei involved in patterned respiratory-vocal activity.     

Origin of projections from the midbrain raphe nuclei to the hypothalamic paraventricular nucleus in the rat: a combined retrograde and anterograde tracing study

A number of neuronal functions governed by the hypothalamic paraventricular nucleus are influenced by serotonin, and it is generally believed that the moderate density of serotonin-immunoreactive fibres and terminals within the paraventricular nucleus originates from the midbrain dorsal and median raphe nuclei. To further evaluate the intricate anatomy of projections from brain stem raphe nuclei of the rat, a combination of retrograde and anterograde tracing experiments were conducted to determine the medullary raphe nuclei projection to the paraventricular nucleus. Rhodamine-labelled latex microspheres, Cholera toxin subunit B and FluoroGold we used as retrograde tracers. Intracerebroventricular injections into the third ventricle of all retrograde tracers labelled a distinct population of neurons in the dorsal raphe situated in the subependymal stratum adjacent to the cerebral aqueduct indicating that these cells take up the tracer from the cerebrospinal fluid. Very few retrogradely labelled neurons were seen in the median raphe after i.c.v. administration of the tracers. Retrograde tracers delivered into the medial part of the paraventricular nucleus labelled no further cells in the midbrain dorsal and median raphe nuclei, whereas a substantial number of retrogradely labelled cells emerged in the pontine raphe magnus. However, when the retrograde tracers were delivered into the lateral part of the paraventricular nucleus, avoiding leakage of the tracer into the ventricle, very few labelled neurons were seen in the dorsal and median raphe, whereas the prominent labelling of raphe magnus neurons persisted. The anatomical organization of nerve fibres terminating in the area of the paraventricular nucleus originating from midbrain raphe nuclei was studied in a series of anterograde tracing experiments using the plant lectin Phaseolus vulgaris leucoagglutinin. Injections delivered into the dorsal raphe or median raphe labelled but a few fibres in the paraventricular nucleus proper. A high number of fine calibered nerve fibres overlying the ependyma adjacent to the paraventricular nucleus was, however, seen after the injections into the subependymal rostral part of the dorsal raphe. Injections delivered into the raphe magnus gave rise to a dense plexus of terminating fibres in the parvicellular parts of the paraventricular nucleus and moderately innervated the posterior magnocellular part of the paraventricular nucleus as well as the magnocellular supraoptic nucleus. Concomitant visualization of serotonin-immunoreactive neurons and retrograde FluoroGold-tracing from the paraventricular nucleus revealed that none of the serotonergic neurons of the raphe magnus projects to this nucleus, while a few of the neurons putatively projecting to the paraventricular nucleus from the median raphe are serotonergic. The current observations suggest that the raphe magnus constitute by far the largest raphe input to the paraventricular nucleus and strongly questions the earlier held view that most raphe fibres innervating the paraventricular nucleus are derived from the midbrain dorsal and median raphe. However, the source of serotonergic innervation of the paraventricular nucleus remains elusive.