Striato-nigral dynorphin and substance P pathways in the rat

Summary In the present study the functional role of the striato-nigral dynorphin and substance P pathways in rat brain has been studied using the rotational behavioural model and an intracerebral dialysis technique complemented with brain lesions and immunohistochemical analysis. Attempts were made to evaluate whether these striato-nigral neurons have a feed-back modulatory action on the dopaminergic nigro-striatal system, or whether they represent an outflow pathway conveying motor information from the striatum. Unilateral injection of dynorphin A into the substantia nigra reticulata of naive rats induced contralateral rotational behaviour. This effect was dose-dependent and mimicked by the kappa-opioid receptor agonist, U50,488H. Intranigral injection of substance P, as well as substance K, also produced dose-dependent contralateral rotational behaviour. Unilateral injections of ibotenic acid into various sites of the striatum were used to destroy the striato-nigral pathways. The lesions produced a depletion of dynorphin- and substance P-like immunoreactivity in the pars reticulata of the substantia nigra ipsilateral to the lesion and markedly affected the behavioural responses to intranigral peptide injections. Dynorphin A more potently induced contralateral rotation in the lesioned compared to naive non-lesioned rats, suggesting development of supersensitivity for this peptide. Substance P on the other hand, was markedly less potent in inducing rotation in lesioned animals. The rotational responses to both dynorphin A and substance P were potentiated by injection of amphetamine 1 h later, suggesting that both peptides act via nigro-striatal dopamine neurons. However, in rats with unilateral nigro-striatal dopamine denervation, produced with 6-hydroxy-dopamine, dynorphin A retained its potency to induce rotational behaviour; substance P was again much less potent. Thus, both the ibotenic acid and 6-hydroxy-dopamine lesions differently affect the action of dynorphin A and substance P in the zona reticulata of the substantia nigra. The data suggests that substance P requires an intact dopamine pathway to produce the rotational response, while dynorphin A does not. Direct evidence that behavioural activation produced by dynorphin A is not dependent upon dopamine stimulation was obtained by intrastriatal dialysis experiments in which changes in striatal dopamine release were measured following intranigral injection of dynorphin A or substance P. Intranigral dynorphin A in fact reduced, while substance P increased the release of dopamine. It is concluded that the dynorphin and substance P striatonigral pathways have different functions. Thus, substance P in the striato-nigral pathway may have a role in a positive feed-back loop regulating the firing of nigro-striatal dopamine neurons, while dynorphin might be important in negative feed-back control. The rotational behaviour produced by DYN A is probably due to direct stimulation of receptors located on nigro-thalamic and nigro-tectal pathways.     

Origins and pathways of cerebrovascular nerves storing substance P and calcitonin gene-related peptide in rat

Origins and pathways of cerebrovascular substance P- and calcitonin gene-related peptide-positive nerves in rat were studied by immunohistochemistry combined with denervation experiments and retrograde axonal tracer technique. The two peptides have been found to coexist in one and the same neuron. After sectioning of the nasociliary nerve bilaterally the substance P/calcitonin gene-related peptide fibers in the rostral half of the circle of Willis and its branches were eliminated, whereas the number decreased in the caudal half of the circle of Willis and rostral two thirds of the basilar artery. Substance P/calcitonin gene-related peptide fibers in the internal carotid arteries, the caudal third of the basilar artery and the vertebral arteries were not affected by the nerve section. After application of the retrograde axonal tracer True Blue onto the proximal segment of the middle cerebral artery the dye accumulated in several Substance P/calcitonin gene-related peptide-containing cells in the ophthalmic division of the ipsilateral trigeminal ganglion and in a few cells in the maxillary trigeminal division and in the internal carotid miniganglion. No other cranial ganglia accumulating the dye contained any substance P/calcitonin gene-related peptide-positive cells. It is concluded that the rostral portion and part of the caudal portion of the cerebral vessels are innervated by substance P/calcitonin gene-related peptide-containing fibers from the trigeminal ganglion and the internal carotid miniganglion. The great majority of trigeminal fibers reach the vessels via the nasociliary nerve of the ophthalmic division, which enters the cranial cavity through the ethmoidal foramen, whereas fibers from the miniganglion project directly to the bypassing internal carotid artery. A probable pathway for the fibers from the maxillary division is suggested. The caudal portion receives, in addition, a supply from other sensory ganglia (lower cranial and/or upper cervical dorsal root ganglia).     

Topography of cholinergic and substance P pathways in the habenulo-interpeduncular system of the rat. An immunocytochemical and microchemical approach

The topography of cholinergic and substance P containing habenulo-interpeduncular projections has been studied in the rat. The research has been carried out by combining choline acetyltransferase and substance P immunohistochemistry to experimental lesions and biochemical assays in microdissected brain areas. In addition, computer-assisted image analysis has been performed in order to obtain quantification of immunohistochemical data. The results show that cholinergic and substance P containing neurons have a different localization in the medial habenula and project to essentially different areas of the interpeduncular nucleus. Cholinergic neurons are crowded in the ventral two-thirds of the medial habenula while substance P containing cells are exclusively localized in the dorsal part of the nucleus. In most parts of the interpeduncular nucleus, choline acetyltransferase and substance P containing fibres and terminals are similarly segregated and no overlapping is apparent except for the rostralmost and the caudalmost ends of the nucleus. Cholinergic activity is largely concentrated in the central core of the nucleus, while substance P is preferentially localized in the peripheral subnuclei of the interpeduncular nucleus. In addition, both substance P and choline acetyltransferase levels show peculiar regional variations along the rostrocaudal axis of the interpeduncular nucleus. The results of experimental lesions demonstrate that the substance P projection carried by each fasciculus retroflexus is prevailingly ipsilateral in the rostral part of the interpeduncular nucleus and becomes progressively bilateral as far as more caudal regions of the nucleus are reached. By contrast, the cholinergic projections carried by each fasciculus retroflexus intermingle more rapidly and only show a slight ipsilateral dominance in the interpeduncular nucleus. The results of the study are discussed with reference to previous anatomical and neurochemical data which, in several instances, had given rise to discrepant interpretations.     

The comparative distribution of enkephalin, dynorphin and substance P in the human globus pallidus and basal forebrain

Three neuropeptides, enkephalin, dynorphin, and substance P appear in the globus pallidus in a unique pattern termed woolly fibers as described previously [Haber and Nauta (1983) Neuroscience 9, 245-260]. The comparative distribution of these fibers are described in the human globus pallidus and basal forebrain area. The results show two main points: The human globus pallidus is a larger, more intricately shaped structure than previously thought, invading several limbic-related basal forebrain regions. There are differences in the distribution patterns of the neuropeptides described, so that they are found in overlapping, but not matching regions. The relationship between the peptide distribution and what is known about the functional (limbic vs motor) circuitry of the region is discussed.     

Immunohistochemical localization of substance P and substance P receptor (NK1) in the olivary pretectal nucleus of the rat

The olivary pretectal nucleus (OPN) is the first central nucleus in the pupillary light reflex arc (PLR). Substance P (SP) is a neuropeptide present in the OPN. The present immunohistochemical study, performed at the ultrastructural level, aimed to determine the synaptic localization of SP and SP receptor in the OPN. Three types of SP-positive terminals were found. The most abundant type was of retinal origin, characterized by electron-lucent mitochondria and round vesicles, organized in glomerular structures, making asymmetric synaptic contacts with dendrites, and profiles containing pleomorphic vesicles, also making synaptic contacts with dendrites. The second type of SP-immunoreactive terminal contained electron-dense mitochondria and pleomorphic vesicles. This type made symmetric synaptic contacts and may originate from the ventral part of the lateral geniculate nucleus. The third type of SP-immunoreactive terminals contained electron-dense mitochondria, clear round vesicles, and made an asymmetric synaptic contact. This type originates from the contralateral OPN. SP receptors of the NK1 subtype were revealed to be on dendrites and were part of the glomerular-like arrangement. On account of the present observations, it can be concluded that retinal projections to the OPN use SP as a neuromodulator and synapse on NK1 receptor-containing dendrites of large neurons projecting to the Edinger-Westphal nucleus. Since SP also modulates the parasympathetic component of the PLR, we postulate that SP plays a modulating role in all components of the PLR.     

Intrathecal somatostatin, somatostatin analogs, substance P analog and dynorphin A cause comparable neurotoxicity in rats.

Rats chronically implanted with intrathecal catheters received intrathecal injections (10 microliters followed by 10 microliters saline flush) of either saline (n = 5), somatostatin (100 micrograms, n = 10), the somatostatin analog BIM 23003 (100 micrograms, n = 5), the somatostatin analog SMS 201-995 (100 micrograms, n = 5), the substance P analog [D-Pro2, D-Trp7,9] SP (10 micrograms, n = 10), or dynorphin A (1-17) (20 nmol, n = 8). These doses (somatostatin, substance P and dynorphin A) were selected based on previous studies in which they caused significant motor deficits. Effects on thermal cutaneous nociception, behavior, motor function and spinal cord histopathology were evaluated. All peptides caused severe neurotoxicity, evidenced by flaccid hind leg paralysis and lumbar spinal neuronal degeneration, which was accompanied by an inflammatory reaction in meninges and spinal gray matter. Histopathological changes had developed within 24 h after injection of somatostatin, substance P analog and dynorphin A, showing mild to severe neuronal degeneration and mild inflammatory responses in spinal cord and meninges. Significant antinociceptive effects, due to severe neurotoxic effects, were only observed following intrathecal injection of SMS 201-995 and the substance P analog. Potential neurotoxic mechanisms of the different peptides are discussed.     

Co-expression of neuropeptides in the cat's striatum: an immunohistochemical study of substance P, dynorphin B and enkephalin.

The expression of tachykinin-like and opioid-like peptides was studied in medium-sized neurons of the caudate nucleus in tissue from adult cats pretreated with colchicine. Two methods, a serial thin-section peroxidase-antiperoxidase technique and a two-fluorochrome single-section technique, were applied. Quantitative estimates were made mainly with the peroxidase-antiperoxidase method. The numbers of neurons expressing substance P-like, dynorphin B-like, and enkephalin-like immunoreactivity were recorded in regions identified, respectively, as striosomes and extrastriosomal matrix. Striosomes were defined by the presence of clustered substance P-positive and dynorphin B-positive neurons and neuropil. Tests for the co-existence of enkephalin-like peptide and glutamate decarboxylase-like immunoreactivity were also made with the peroxidase-antiperoxidase method. Co-expression of substance P-like and dynorphin B-like immunoreactivities was the rule both in striosomes and in the matrix. In striosomes, substance P-like immunoreactivity was found in 96% of dynorphin B-immunoreactive neurons, and in the matrix 89% of dynorphin B-positive cells contained substance P-like immunoreactivity. Substance P/dynorphin B-positive neurons corresponded to over half (57%) of the neurons in striosomes but only 39% of the neurons in the matrix. Both in the matrix and in striosomes, about two-thirds of all neurons (63% and 65%, respectively) were identified as enkephalin-positive. Among all substance P/dynorphin B-positive medium-sized neurons, 76% also contained enkephalin-like antigen. The enkephalin-positive neurons characterized by triple peptide co-existence (enkephalin/substance P/dynorphin B) represented a mean of 63% of striosomal enkephalin-positive neurons (41% of all striosomal neurons) and 35% of matrical enkephalin-positive neurons (26% of all matrical neurons). Finally, nearly all enkephalin-positive neurons were immunoreactive for glutamate decarboxylase, and therefore probably GABAergic, but only about half the glutamate decarboxylase-positive population was enkephalin-immunoreactive. These findings suggest that neuropeptides from three distinct precursors may be co-localized in single medium-sized neurons in the striatum, and that the differential patterns of co-expression of substance P-like, dynorphin B-like, and enkephalin-like peptides may confer functional specializations upon subpopulations of GABAergic neurons giving rise to the efferent projections of the striatum. The linked expression of substance P-like and dynorphin B-like peptides in single neurons both in striosomes and matrix suggests that some regulatory mechanisms controlling peptide expression apply regardless of compartment.(ABSTRACT TRUNCATED AT 400 WORDS)     

The substance P innervation of the rat hippocampal region

Summary The distribution of substance P (SP) immunoreactive nerve cell bodies and preterminal processes was studied in the rat brain by using several anti-SP-antibodies in combination with immunohistochemical techniques. In normal rats and in rats pretreated with colchicine, SP immunoreactive preterminal processes were found in the hippocampal region, but SP positive cellbodies could be detected only after colchicine pretreatment. Medium-sized to large, multipolar cells immunoreactive for SP were found in stratum oriens of the hippocampal subfield CA3 and in the hilus of the area dentata. Medium-sized to small, round or fusiform cells were detected in the pyramidal layer of the ventral subiculum and in layers III–VI of the ventral entorhinal area. The SP stained preterminal processes were of two types. Numerous fine, varicose axons were stained in different parts of Ammon's horn, while in the retrohippocampal structures, the SP immunoreactivity was present in small distinctly stained puncta. These frequently formed pericellular arrangements around unstained cells, indicative of axosomatic contacts between SP terminals and cells in the hipocampus. In Ammon's horn, the densest SP innervation was found in strata oriens, radiatum and moleculare of subfields CA3a and CA2. Scattered fibers were also present in the stratum oriens of CA3a-c and in the hilus, in particular at ventral levels. In retrohippocampal structures, the SP innervation predominated in the deep pyramidal layer of the subiculum, the second layer of the presubiculum and in layers VI and IV of the medial and lateral entorhinal area. Many of these terminals may arise from local interneurons as well as from sources outside the hippocampal region.Taken together, these studies demonstrate a far more extensive innervation by SP, or a closely related peptide, of the rat hippocampal region than was previously recognized. This suggests that SP may play an important role in neurotransmission within the hippocampal region.Stephen Davies was supported by Travel grants from the Wellcome Trust and the Gurantors of Brain.     

Activation by stress of the habenulo-interpeduncular substance P neurons in the rat

Substance P levels were significantly decreased in the ventro-tegmental area and the interpeduncular nucleus of rats submitted for 20 min to electric foot-shocks. Substance P levels in the substantia nigra and in the prefrontal cortex were not affected. These results are discussed in light of the selective activation of the mesocortico-prefrontal dopaminergic neurons induced by this stress situation.     

Electrophysiological effects of substance P and VIP in the denervated rat parotid gland

The effects of SP and VIP were studied in vitro in normal and denervated parotid glands of the rat using electrophysiological techniques. After sympathetic, and especially after parasympathetic, denervation the number of cells responding to peptides increased significantly. Furthermore, sub-threshold doses for control glands induced membrane potential changes after denervation. It seems reasonable to correlate these findings to the development of supersensitivity. The underlying mechanisms, however, require further investigation.     

Ultrastructural localization and afferent sources of substance P in the rat parabrachial region

The ultrastructural morphology and afferent sources of terminals containing substance P-like immunoreactivity were examined in the rat parabrachial region. In the first portion of the study, a polyclonal antiserum to substance P was localized in the ventrolateral parabrachial region using the peroxidase-antiperoxidase labeling technique combined with electron microscopy. The antiserum was tested for cross-reaction with substance P, physalaemin, substance K and neuromedins B, C and K. Cross-reactivity was most intense with substance P. However, substance K, neuromedin K and physalaemin also exhibited limited cross-reactions with the antiserum. In the ventrolateral parabrachial region of untreated adult animals, substance P-like immunoreactivity was localized in axon terminals containing numerous small (40-60 nm) clear vesicle and 1-3 large (90-120 nm) dense-core vesicles. At least 54% of the labeled terminals formed asymmetric synapses with unlabeled dendrites; and at least 30% of the recipient dendrites received more than one labeled axon terminal. In addition, the labeled terminals were associated less frequently with other unlabeled soma, axon terminals and blood vessels. In the second part of the study, we examined whether or not perikarya in various extrinsic regions contributed to the substance P-like immunoreactivity in axon terminals in the parabrachial region. Wheat-germ agglutinin conjugated horseradish peroxidase was injected unilaterally into the parabrachial region of adult rats two days prior to being killed and one day prior to intraventricular injection of colchicine (100 micrograms in 7.5 microliter saline) which enhanced the detection of immunoreactivity in perikarya. Sections were first processed by a tetramethylbenzidine reaction stabilized with cobalt-diaminobenzidine for demonstration of the transported peroxidase then were immunocytochemically labeled for substance P. Perikarya containing both the black granular retrograde labeling and brown peroxidase-immunoreactivity were found in the nuclei of the solitary tracts, the caudal ventrolateral reticular formation, the lateral dorsal tegmental nucleus and the paraventricular, dorsomedial and lateral hypothalamic nuclei. The projections were largely, but not exclusively, from perikarya located on the same side as the parabrachial injection. We conclude that substance P, or a closely related tachykinin, is a putative transmitter or modulator within a number of pathways to the parabrachial region and that these afferents act primarily through axodendritic synapses with intrinsic neurons.     

Cholecystokinin and substance P immunoreactive projections to the paraventricular thalamic nucleus in the rat

Cholecystokinin (CCK) and substance P (SP) are thought to play an important role in a variety of stress responses. Both CCK- and SP-positive fibers innervating the thalamus are found principally in the midline nuclei, including the paraventricular thalamic nucleus (PVT), which has strong reciprocal connections with the medial prefrontal cortex. In the present study, we determined the source of the CCK- and SP-immunoreactive fibers to the PVT, employing combination of retrograde neuronal tracing and immunohistochemistry in the rat. The PVT-projecting neurons showing CCK immunoreactivity were detected in the dorsomedial nucleus of the hypothalamus, and ventral mesencephalic periaqueductal gray, including the Edinger-Westphal nucleus and the dorsal raphe nucleus. Sources of SP afferents to the PVT were detected in the Edinger-Westphal nucleus, the mesopontine tegmentum and the medullary raphe nucleus. CCK- and SP-immunoreactive fibers may exert modulatory influence on the prefrontal cortical activity via the PVT and regulate behavioral components of stress-adaptation responses.     

Temporal changes in spinal cord expression of mRNA for substance P,dynorphin and enkephalin in a model of chronic pain

We have used a partial sciatic nerve ligation model to examine the time course for changes in the expression of mRNA for three peptides related to pain transmission at spinal sites (dynorphin, enkephalin and substance P), during the development of allodynia. Enhanced expression of mRNA for dynorphin and substance P was observed in the dorsal horn on the same side as the partial nerve ligation. Increased expression of dynorphin mRNA was biphasic. The initial increases in expression of dynorphin mRNA occurred at 3 h, and a secondary peak was observed 1–3 days after surgery. The secondary increases coincided roughly with increased substance P mRNA expression. However, both dynorphin and substance P mRNA returned to control values after 1 week despite continuing allodynia. No significant changes in expression of mRNA for enkephalin were observed. The elevation of substance P mRNA in intrinsic spinal cord neurons may be secondary to changes in immediate early genes c-fos and jun-B, whereas the expression of dynorphin and enkephalin mRNA is differently regulated. The results also suggest that changes in the expression of the three neuropeptides are not critically involved in the development and maintenance of chronic pain or allodynia.     

In situ hybridization analysis of substance P receptor in the rat retina

Substance P receptor is known to provide a principal interface between tachykinin peptides and tachykinin-sensitive cells in retinal circuitry and to produce several physiological functions such as excitation of ganglion cells. We reported results of in situ hybridization analysis of substance P receptor in rat retina using digoxigenin-labeled RNA probes to yield discrete cell labeling. Distinct hybridization signal was present in a great majority of ganglion cells that provide retinal fibers to a central target. It was also present in a subpopulation of amacrine cells. Following optic nerve crush, ganglion cells lost their hybridization signal in a time-dependent manner, while hybridization-positive amacrine cells were persistently seen. From the results, we identified the hybridization message as distinctly localized to two systems, output cells and intrinsic cells in retinal circuitry.     

Neural actions of several substance P antagonists in the rat spinal cord

Four substance P (SP) antagonists were tested on anaesthetized rats, by injecting 8 microgram amounts into the spinal cord (T8-T9), and by observing their effects on the hypothalamo-neurohypophysial responses to a presumably painful stimulus, the superfusion of the hepatic portal vein with 1 microM bradykinin. Only two antagonists, the new D-Pro4,D-Trp7,9,10,Val8-SP4-11 and D-Pro4,D-Trp7,9,10-SP4-11 were capable of inhibiting the responses by 50-60%, the former compound having 3 times less agonistic activity. The results suggest that substitution of the aromatic phenylalanine by the non-polar valine in position 8 may significantly improve the overall characteristics of neurally active SP antagonists.     

Immunohistochemical observation on substance P in regenerating taste buds of the rat

The present study was performed to investigate the relationship between substance P-positive (SP-positive) nerve fibers and regeneration of taste buds in the foliate papillae of the rat by means of immunohistochemistry. It was confirmed by neurotomy that taste buds in the foliate papillae of the rat were innervated mostly (90%) by the glossopharyngeal (IXth) nerve and partly (10%) by the chorda tympani. In this experiment, the IXth nerve was sectioned distal to the petrosal ganglion. The rats were sacrificed at various intervals from 20 days to 80 days after the operation. In the course of degeneration and disappearance of taste buds, both SP-positive fibers and taste buds disappeared completely from the posterior folds of the foliate papillae 7 days after the operation. Within 22 days, regenerated SP-positive fibers began to appear in the lamina propria, and following the penetration of the fibers into the epithelium, taste bud anlagen reappeared at the bottom of the trench, and in the posterior folds at 24 days. The process of new taste bud formation extended toward the apex of the trench and to the anterior folds, which seemed to follow the regeneration in the nerve trunk. Quantitative data showed a gradual increase in the number of taste buds and taste buds containing SP-positive fibers. These findings indicate that SP might have a role in regeneration of taste buds.     

Role of dynorphin and enkephalin in the regulation of striatal output pathways and behavior

Projection neurons in the striatum give rise to two output systems, the “direct” and “indirect” pathways, which antagonistically regulate basal ganglia output. While all striatal projection neurons utilize GABA as their principal neurotransmitter, they express different opioid peptide co-transmitters and also different dopamine receptor subtypes. Neurons of the direct pathway express the peptide dynorphin and the D1 dopamine receptor, whereas indirect pathway neurons express the peptide enkephalin and the D2 receptor. In the present review, we summarize our findings on the function of dynorphin and enkephalin in these striatal output pathways. In these studies, we used D1- or D2-receptor-mediated induction of immediate-early genes as a cellular response in direct or indirect projection neurons, respectively, to investigate the role of these opioid peptides. Our results suggest that the specific function of dynorphin and enkephalin is to dampen excessive activation of these neurons by dopamine and other neurotransmitters. Levels of these opioid peptides are elevated by repeated, excessive activation of these pathways, which appears to be an adaptive or compensatory response. Behavioral consequences of increased opioid peptide function in striatal output pathways may include behavioral sensitization (dynorphin) and recovery of motor function (enkephalin).     

Desensitization of rat peritoneal mast cells to substance P

Rat peritoneal mast cells were pretreated for 10 min at 37°C with either substance P (SP, 3 or 6 M) or compound 48/80 (37.5, 50 or 75 ng/ml). The effect of this pretreatment on the subsequent responsiveness of the cells to SP was studied. Both SP and compound 48/80 pretreatment of rat peritoneal mast cells inhibited the subsequent response of the cells to SP. The degree of inhibition produced by either SP or compound 48/80 was dependent on the concentration used to pretreat the cells. Inhibition of the response of the cells to SP was observed whether or not the pretreating agent was removed or remained in contact with the cells during the subsequent stimulation with SP. It is concluded that compound 48/80 and SP desensitize the cells to subsequent stimulation by SP and possible mechanisms for this are discussed.