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Bradykinin B2 receptors were localized in the sheep brain and spinal cord by quantitative in vitro autoradiography using a radiolabelled and specific bradykinin B2 receptor antagonist analogue, 3-4-hydroxyphenyl-propionyl-D-Arg0-[Hyp3,Thi5,D-Tic 7,Oic8]bradykinin, (HPP-HOE 140). This radioligand displays high affinity and specificity for bradykinin B2 receptors. The respective K(i) values of 0.32, 1.37 and 156 nM were obtained for bradykinin, HOE140 and D-Arg[Hyp3,D-Phe7,Leu8]bradykinin competing for radioligand binding to lamina II of sheep spinal cord sections. Using this radioligand, we have demonstrated the distribution of bradykinin B2 receptors in many brain regions which have not been previously reported. The highest density of bradykinin B2 receptors occur in the pleoglial periaqueductal gray, oculomotor and trochlear nuclei and the circumventricular organs. Moderate densities of receptors occur in the substantia nigra, particularly the reticular part, the posterior thalamic and subthalamic nuclei, zona incerta, the red and pontine nuclei, some of the pretectal nuclei and in discrete layers of the superior colliculus. In the hindbrain, moderate levels of bradykinin B2 receptor binding occur in the nucleus of the solitary tract, and in spinal trigeminal, inferior olivary, cuneate and vestibular nuclei. Laminae II, X and dorsal root ganglia display the most striking binding densities in the spinal cord, while the remainder of the dorsal and ventral horn display a low and diffuse density of binding. Bradykinin B2 receptors are extensively distributed throughout the sheep brain and spinal cord, not only to sensory areas but also to areas involved in motor activity.

The anatomical arrangement of catecholaminergic nerve terminals in the intermediate zone of the thoracolumbar spinal cord was examined with the fluorescence microscope in serial sections of spinal cord from adult cats perfused with a formaldehyde-glutaraldehyde mixture. Specific fluorescence in this material was assumed to represent noradrenaline. The distribution of fluorescent varicose nerve terminals was compared with that of neuron cell bodies in the same sections after Nissl counterstaining, and with the known topographic distribution of sympathetic preganglionic neurons. Dense accumulations of noradrenergic fibers were found in the intermediate zone in all segments between the caudal part of C8 and the rostral part of L4. These were not only associated with preganglionic neurons in the intermediolateral columns (ILN), but below T5 also extended in bands (1-3 per mmm of spinal cord) toward, and in some segments across, the midline. Noradrenergic terminals were associated with cell bodies in most parts of the ILN from T1 to T8 and in L2-3. Between T9 and L2, the innervation of the ILN was patchy, and the majority of the noradrenergic fibers projected to regions medial to the ILN. These corresponded to sites at which preganglionic neurons and also possible interneurons of the intermediomedial nucleus occur. Although preganglionic neurons and clusters of noradrenergic terminals are located in similar regions across the intermediate zone, their densities and patterns of distribution differ. This observation applies to comparisons both between anatomical subnuclei and between segments in a characteristic way along the length of the thoracolumbar cord.

Immunocytochemical procedures have been used to examine the distributions of substance P (SP)-positive fibres within the intermediate zone of the thoracolumbar spinal cords of rabbits, cats, and monkeys. In all three species SP fibres were concentrated in areas known to contain sympathetic preganglionic neurones. These included the intermediolateral nucleus and the funiculus just lateral to it, the medial gray matter in the area of the nucleus intercalatus, and the paracentral region. The density of the SP innervation varied in a characteristic way both between these subpopulations of sympathetic neurones and in its overall input to different segmental levels. Generally the greatest accumulations of SP fibres were found in the T3-T5 and L2-L4 regions and these were concentrated in the intermediolateral nucleus (ILN). The highest densities of SP fibres in the lateral funiculus were in the upper thoracic and upper lumbar segments whereas SP fibres forming transverse bands, possibly in association with neurones in the nucleus intercalatus, were most prominent in T5-T8. Substance P fibres adjacent to the midline were more or less equally dense throughout the segments examined. Substance P-positive cell bodies situated immediately lateral to the central canal were present at a density of 200-300 per segment throughout the cat thoracolumbar cord. These neurones may be the cells of origin of at least some of the SP fibres in the intermediate zone. The close association of sympathetic preganglionic neurones with SP fibres, many of which are thought to be derived from cells in the medulla, suggests a role for SP-containing fibres in the modulation of sympathetic activity. The variation in input to different segments and classes of sympathetic neurones further suggests a specificity which may be related to the different functions of the neurones innervated.

The distribution of sensory cells whose axons traverse the stellate ganglion and project via sympathetic cardiac nerves to the heart of the cat has been examined quantitatively. Horseradish peroxidase (HRP) injected at multiple sites in the right stellate ganglion, or applied to the middle cardiac nerve, labelled small numbers of cells in the thoracic dorsal root ganglia (DRG) from T1 to T8. These cells were most numerous between T2 and T5 and were consistently small (less than 40 micrometer) relative to other cells in the DRG. When HRP was applied to middle cardiac nerves, the numbers of labelled sensory cells always exceeded the numbers of myelinated axons counted in the same nerves from other cats. It is concluded that the distribution of the cells of cardiac sensory fibers is more extensive within thoracic DRG than has been previously reported, and it is suggested that such fibres travelling in the sympathetic cardiac nerves may be either myelinated or unmyelinated.

The distribution of sympathetic preganglionic neurons that project via the right stellate ganglion has been studied quantitatively in adult cats. Retrograde transport of horseradish peroxidase (HRP) injected into the ganglion or applied to transected axons of the cervical sympathetic trunk (CST) resulted in labelling of neurons in the ipsilateral spinal cord over T1-T9 and T1-T7, respectively. Their distribution and morphology in the subnuclei of the intermediate zone were determined. Neurons within the principal part of the intermediolateral column (ILp) comprised the majority of labelled cells at all levels, irrespective of the site of HRP application, while more medially located neurons projected differentially. A combination of the application procedures labelled what appeared to be a sum of the individual projections, and few ILp cells remained unlabelled in the most rostral segments. From reconstructions of different segmental levels, most of the cells in the ILp were found to lie in a column approximately 200 micrometer in diameter composed of a series of cell aggregations of 20-150 neurons at intervals of approximately 300 micrometer. Less frequently, mediolaterally aligned cells extended toward the central canal, near which small cell clusters were also detected. After selective HRP application to individual rami of T1-T3, labelled cells were restricted to one segment's length, so that neurons located more caudally must project extraspinally to the stellate ganglion. Consideration of the maximum cell numbers labelled by each procedure suggests that preganglionic collaterals (probably unmyelinated) diverging from the pathway to the preferred target of their parent neuron were not labelled by this technique.

Light microscopic studies in our laboratory have indicated that the lateral septum, amygdala, and ventral subiculum project in a perinuclear fashion to the paraventricular (PVN), supraoptic (SON), and suprachiasmatic (SCN) nuclei (Oldfield et al., '82; Silverman and Oldfield, '84). In the present paper a combined anterograde HRP and immunocytochemical procedure has been used to determine the connectivity between these limbic efferents and peptide-containing processes emanating from the above mentioned hypothalamic nuclei. Synaptic associations were found to exist between efferents from (1) the septum and both vasopressin (VP)- and oxytocin (OX)-positive dendrites derived from cells in the PVN and SON, (2) the septum and VP dendrites dorsal to the SCN, (3) the ventral subiculum and both VP and OX dendrites arising from the PVN and SON, and (iv) the amygdala and VP dendrites from the PVN. These observations help clarify an apparent discrepancy between electrophysiological data, in which limbic efferents have been shown to influence the activity of VP and OX neurons in the PVN and SON, and anatomical evidence which indicates only a perinuclear innervation from these sites not encroaching on the hypothalamic nuclei themselves. In each case the synaptic connections are made on dendrites external to the nucleus: those lateral and ventrolateral to the PVN, dorsal to the SON, and dorsal or dorsolateral to the SCN.