Demonstration of glutamate immunoreactivity in vagal sensory afferents in the nucleus tractus solitarius of the rat.

To investigate whether glutamate is a neurotransmitter in vagus nerve sensory afferents terminating in the nucleus tractus solitarius, these terminals were identified by the anterograde transport and their glutamate content examined using the post-embedding immunogold technique. After injection of horseradish peroxidase into the nodose ganglion anterogradely labelled axonal boutons were visualized throughout the nucleus of the solitary tract (nTS), the dorsal motonucleus of the vagus nerve (DVN), predominantly ipsilateral to the injection, and to a lesser extent in the area postrema. Electron microscopic analysis of 47 anterogradely labelled boutons in the nTS following post-embedding immunocytochemistry for glutamate revealed that 43 of these boutons (> 91%) contained a level of glutamate immunoreactivity significantly greater (P < 0.001%) than that observed in the surrounding tissue. The observed enrichment of glutamate immunoreactivity in boutons identified as vagus nerve sensory afferents indicate that glutamate may be a transmitter in these neurones.

Responses of ventral respiratory neurones in the rat to vagus stimulation and the functional division of expiration.

In anaesthetized rats, extracellular and intracellular recordings were taken from 106 respiratory neurones in the intermediate region of the nucleus ambiguus. We observed unprovoked shortening of expiratory time accompanied, in all classes of respiratory neurone, by the elimination of the changes in membrane potential that were characteristic of stage II expiration. The demonstration of the elimination of stage II expiration in both the rat and cat strongly supports the functional division of expiration into stage I expiration (post-inspiration) and stage II expiration. In order to identify the neurones in the rat that receive inputs from vagal afferents and modulate the central respiratory rhythm, we examined whether any respiratory neurones responded to stimulation of the vagus nerve. Some post-inspiratory and stage II expiratory neurones responded. The short latency (< 2 ms) of four of the responses indicates that some vagal afferents act on post-inspiratory neurones via a disynaptic pathway. While repetitive stimulation of the vagus nerve could inhibit the phrenic rhythm, it appears that most inspiratory neurones in the intermediate region of the nucleus ambiguous complex are not directly involved in integrating the information from vagal afferents with the central respiratory rhythm.

Central distribution of substance P, calcitonin gene-related peptide and 5-hydroxytryptamine in vagal sensory afferents in the rat dorsal medulla.

The central distribution of vagal afferents in the medulla containing either substance P, calcitonin gene-related peptide or 5-hydroxytryptamine was examined using a double-labelling technique and laser scanning confocal microscopy. Areas of the nucleus tractus solitarii, dorsal motonucleus of the vagus nerve and area postrema were scanned for double-labelled axon profiles. Analysis of this material revealed that all three neurochemicals were contained within the central terminals of vagal nerve sensory neurons. However, the distribution of vagal nerve afferents containing each of these putative transmitters differed. Afferents containing 5-hydroxytryptamine were detected mainly in the areas postrema and the adjacent nucleus tractus solitarii, with a smaller number in the ventral subnuclei of the solitary tract. In contrast afferents containing calcitonin gene-related peptide were found primarily in the medial and commissural regions of the nucleus tractus solitarii. Afferents containing substance P-immunoreactivity were surprisingly few in number and did not appear to be associated with any particular region. These results establish the presence of 5-hydroxytryptamine, substance P and calcitonin gene-related peptide in the central axons of vagal sensory afferents. Furthermore, the differential distribution of afferents immunoreactive for these neurochemicals seen in this study, together with previous demonstrations of the viscerotopic organization of vagal sensory afferents suggests a possible "chemical coding" for individual end organs.

Localization of cardiac vagal preganglionic motoneurones in the rat: immunocytochemical evidence of synaptic inputs containing 5-hydroxytryptamine.

The origin of cardiac vagal preganglionic motoneurones in the rat is still controversial and knowledge of the chemistry of synaptic inputs onto these neurones is limited. In this investigation vagal preganglionic motoneurones innervating the heart were identified by the retrograde transport of cholera toxin conjugated to horseradish peroxidase (CT-HRP) combined with the immunocytochemical localization of 5-hydroxytryptamine. Injection of CT-HRP into the myocardium resulted in the retrograde labelling of neurones primarily in the ventral regions of the nucleus ambiguus (75.1%). Labelled neurones were also distributed in a narrow band through the reticular formation extending between the dorsal motor nucleus of the vagus nerve and the nucleus ambiguus (17.3%) as well as in the dorsal motor nucleus itself (7.6%). A combination of retrograde labelling with immunocytochemistry for 5-hydroxytryptamine revealed that the neuronal perikarya and the dendrites of cardiac vagal motoneurones in the nucleus ambiguus were often ensheathed in 5-hydroxytryptamine-immunoreactive axonal boutons. Electron microscopic examination of this material confirmed that there were synaptic specializations between these boutons and the cardiac vagal motoneurones. The identification of 5-hydroxytryptamine-containing synaptic inputs to this population of vagal motoneurones provides further detail towards the understanding of the regulation of heart rate by the parasympathetic nervous system.

gamma-Aminobutyric acid immunoreactive structures in the nucleus tractus solitarius: a light and electron microscopic study.

gamma-Aminobutyric acid immunoreactive perikarya and boutons in the nucleus tractus solitarius of the cat were examined at both the light and electron microscopic level. Immunoreactive neurones were found predominantly in the parvocellular subdivision of the nucleus tractus solitarius and to a lesser degree in all the other subdivisions of the nucleus tractus solitarius and the dorsal vagal motonucleus. All the immunoreactive perikarya observed were similar in size and morphology. gamma-Aminobutyric acid immunoreactive boutons were observed throughout the nucleus tractus solitarius. However, in contrast to its high content of immunoreactive perikarya the parvocellular subdivision contained the lowest density of immunoreactive boutons. Ultrastructural examination of immunoreactive boutons in the different regions of the nucleus tractus solitarius revealed that they formed synaptic specializations, predominantly with dendritic shafts, all of which were of the symmetric type. This pattern of innervation was observed throughout the medial, commissural, ventrolateral and parvocellular subdivisions of the nucleus tractus solitarius.

A note on the use of biocytin in anterograde tracing studies in the central nervous system: application at both light and electron microscopic level.

A new neuroanatomical method based on the anterograde transport of biocytin has recently been reported. The validity of this method is examined closely on a number of well established pathways in the central nervous system, as well as the experimental parameters necessary for its effective use. iontophoretic application of this amino acid-biotin complex allows the placement of very discrete injections. At the injection sites neurones appear to be completely filled, whereas fibres of passage are left unlabelled. From the injection site axons can be traced at the light microscopic level to their terminal fields, where their pattern of termination and morphology can be clearly visualised. These anterogradely labelled fibres can be examined further by electron microscopy to identify synaptic specializations. The uptake and transport of biocytin, following iontophoretic injection, appears to be similar to that observed using techniques employing radiolabelled amino acids or Phaseolus vulgaris-leucoagglutinin (PHA-L), in that it is transported predominantly in the anterograde direction. However, biocytin offers some advantages over these techniques in that its detection is relatively easy at electron microscopic levels. Furthermore, biocytin appears to be transported rapidly making it possible to study short pathways under acute experimental conditions.

Cholinergic synaptic input to different parts of spiny striatonigral neurons in the rat.

The postsynaptic targets of cholinergic boutons in the rat neostriatum were assessed by examination in the electron microscope of boutons that were immunoreactive for choline acetyltransferase, the synthetic enzyme for acetylcholine. These boutons formed symmetrical synaptic specializations with neostriatal neurons. Of 209 immunoreactive synaptic boutons observed in random searches of the neostriatum, 45% made contact with dendritic shafts, 34% with dendritic spines, and 20% with neuronal perikarya. Many of the postsynaptic structures had ultrastructural characteristics of the most common type of striatal neuron, the medium-size densely spiny neuron. This was confirmed by the examination in the electron microscope of Golgi-impregnated medium-size spiny neurons from sections that had also been immunostained for choline acetyltransferase. Immunoreactive boutons formed symmetrical synaptic specializations with all parts of the neurons examined, i.e., perikarya, proximal and distal dendritic shafts, and dendritic spines. Two of the Golgi-impregnated medium-size spiny neurons that received input from the cholinergic boutons were also retrogradely labelled with horseradish peroxidase that had been injected into the substantia nigra, they were thus further characterized as striatonigral neurons. Similarly, seven retrogradely labelled perikarya of striatonigral neurons were found to receive input from the cholinergic boutons. It is concluded that cholinergic boutons in the neostriatum form synaptic specializations and that one of their major targets is the medium-size densely spiny neuron that projects to the substantia nigra. The topography of the cholinergic afferents of these cells is distinctly different from that of other boutons derived from local neurons and from boutons that form asymmetrical synaptic specializations, but it is similar to that of the dopaminergic boutons originating from neurons in the substantia nigra.

Cellular substrate of the histochemically defined striosome/matrix system of the caudate nucleus: a combined Golgi and immunocytochemical study in cat and ferret.

In order to learn what morphological substrate might underly the histochemical compartments of the neostriatum, sections of the caudate nucleus and the putamen of cats and ferrets were stained immunocytochemically with antisera directed against several neuropeptides and transmitter-related enzymes and were then Golgi-impregnated. Adjacent sections were stained to reveal acetylcholinesterase activity to identify the acetylcholinesterase-poor striosomes. The immunostaining produced by several of the antibody preparations was in register with the acetylcholinesterase-poor striosomes but the most prominent staining of these zones occurred with the antibodies directed against substance P. The striosomes were delineated by intense substance P-immunostaining of neuronal perikarya and dendrites, and in the rostral and dorsal caudate nucleus the boundary between substance P-immunostained and extrastriosomal matrix was abrupt. For these reasons we analysed Golgi-impregnated neurons in sections immunostained for substance P in order to assess the influence of the chemically defined striosomal architecture on the position and dendritic arborization of neurons located both within the striosomes and within the extrastriosomal matrix. The most commonly impregnated neurons were of the medium-size densely spiny class. Those that were present within the striosomes and lay within one dendritic radius of the boundary were divided into two types: (1) neurons whose dendritic arborization was apparently not influenced by the boundary and (2) neurons whose dendritic arborization was markedly influenced by the boundary. For neurons of the latter type, dendrites either emerged from the parts of the perikaryon away from the boundary, so avoiding crossing it, or they exhibited abrupt changes in their course, apparently to avoid crossing the boundary. Spiny neurons located in the extrastriosomal matrix but close to the striosomal boundary had dendrites that were either influenced by, or not influenced by the compartmental boundary. We conclude that there is a specific cytoarchitecture underlying the histochemical compartments of the neostriatum and that different sub-populations of medium-size spiny neurons underly (1) the segregation of information flow in striosomes and the extrastriosomal matrix and (2) communication between striosomes and the extrastriosomal matrix.

The postsynaptic targets of substance P-immunoreactive terminals in the rat neostriatum with particular reference to identified spiny striatonigral neurons.

Substance P-immunoreactive boutons were examined in the electron microscope in sections of the rat neostriatum that contained retrogradely labelled striatonigral neurons and/or Golgi-impregnated medium-size densely spiny neurons. The post-synaptic targets of the immunoreactive boutons were characterized on the basis of ultrastructural features, their projection to the substantia nigra and/or their somato-dendritic morphology. Substance P-immunoreactive axonal boutons formed symmetrical synaptic specializations. Of a total of 233 randomly identified synaptic boutons 72.5% made contact with dendritic shafts, 15% with dendritic spines and 10.7% with perikarya. The ultrastructural characteristics of some of the postsynaptic neuronal perikarya were consistent with their identification as striatal interneurons. Similarly, the observation of some of the substance P-containing terminals in contact with spines, spine-bearing dendritic shafts and perikarya with the ultrastructural characteristics of medium-size densely spiny neurons suggested that one of the targets of substance P-positive terminals are striatal projection neurons. Direct evidence for this was obtained in sections from rats that had received injections of horseradish peroxidase conjugated with wheatgerm agglutinin in the substantia nigra. The perikarya of retrogradely labeled striatonigral neurons were found to receive symmetrical synaptic input from substance P-positive boutons. Ultrastructural analysis of Golgi-impregnated medium-size densely spiny neurons, some of which were also retrogradely labeled from the substantia nigra, demonstrated directly that this class of neuron was postsynaptic to the substance P-immunoreactive boutons. The combination of Golgi-impregnation with substance P-immunocytochemistry made it possible to study the pattern or topography of the substance P-positive input to medium size densely spiny neurons. The substance P-containing boutons made contact predominantly with perikarya and dendritic shafts. This pattern of input is markedly different from that of other identified inputs to medium-size densely spiny neurons.

Characterization of substance P- and [Met]enkephalin-immunoreactive neurons in the caudate nucleus of cat and ferret by a single section Golgi procedure.

Modifications of the single-section Golgi-impregnation procedure of Gabbott and Somogyi are described. The modifications allow easier and more rapid preparation of the sections for Golgi-impregnation and easier handling of large numbers of serial sections. The technique consists of placing a section that has been treated with osmium tetroxide and potassium dichromate on a microscope slide and "sandwiching" it with a second microscope slide. The two slides are held together at one end by tape and the assembly is dipped into a solution of silver nitrate. Golgi-impregnation of neurons occurs within a few hours and is generally complete within 12 h. The technique has been applied to sections through the caudate nucleus of the cat and ferret in order to define the morphological characteristics of striatal substance P- and methionine enkephalin-immunoreactive neurons. Sections were first incubated to reveal the immunoreactive structures and then subjected to the Golgi method. Golgi-impregnated neurons that were immunoreactive for either substance P or methionine enkephalin had medium-size perikarya from which several dendrites emerged. The dendrites branched close to the perikaryon; secondary and higher order dendrites were densely laden with spines, as many as 15 spines per 10 microns of dendrite. It is concluded that both striatal substance P-containing and methionine enkephalin-containing neurons are of the medium-size densely spiny type. Medium-size densely spiny neurons may be homogeneous with respect to their somatodendritic morphology but heterogeneous with respect to their chemical characteristics and axonal morphology.

Substance P-containing terminals in synaptic contact with cholinergic neurons in the neostriatum and basal forebrain: a double immunocytochemical study in the rat.

Antibodies against substance P and choline acetyltransferase (ChAT) have been used in a sequential double-immunocytochemical ultrastructural study of the rat forebrain. The peroxidase-anti-peroxidase procedure was used for both antigens, however, two different substrates for the peroxidase reactions were used. The substance P-immunoreactive sites were first localized using 3,3'-diaminobenzidine as the substrate, then the ChAT-immunoreactive sites were localized using benzidine dihydrochloride. The reaction product formed by the two substrates was distinguishable in both the light and electron microscopes. Using this procedure, the cell bodies and proximal dendrites of identified cholinergic neurons in the neostriatum were found to receive symmetrical synaptic input from substance P-immunoreactive boutons. A similar pattern of substance P-immunoreactive synaptic input was observed onto magnocellular basal forebrain cholinergic neurons in the ventral pallidum and ventromedial globus pallidus. In both the striatum and basal forebrain substance P-immunoreactive boutons were also seen in contact with structures that did not display ChAT immunoreactivity.