Immunocytochemical distribution of neuropeptide F (NPF) in the gastropod mollusc,Helix aspersa,and in several other invertebrates

The distribution of neuropeptide F (NPF) immunoreactivity in the snail, Helix aspersa, has been demonstrated by immunocytochemistry using 2 regionspecific antisera. One, designated NPF3, was raised against a synthetic N-terminal fragment of Helix aspersa NPF; the other, designated PP221, was raised against the C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP) but cross-reacts fully with the analogous C-terminal region of Helix aspersa NPF. The distribution of NPF immunoreactivity has also been compared with that of FMRFamide using alternate serial sections of Helix aspersa ganglia. Results showed that NPF immunoreactivity was abundant and widespread in the central and peripheral nervous systems and the pattern of immunostaining obtained using both region-specific antisera was similar. Likewise, immunocytochemistry of neural tissues of a congeneric species, Helix pomatia, and 2 prosobranch gastropods, Buccinum undatum and Littorina littorea, produced similar staining patterns with both antisera. However, in the cephalopod mollusc, Loligo vulgaris, and the cestode, Moniezia expansa, positive immunostaining was only obtained with the C-terminal PP antiserum. Immunostaining of alternate serial sections of Helix aspersa ganglia with NPF3, and an antiserum raised to FMRFamide, showed that while a few neurones were immunoreactive with one antiserum only, in the majority, both immunoreactivities were co-localised. NPF thus appears to be an important neuropeptide of widespread distribution in Helix aspersa and the differential immunocytochemical staining obtained using the 2 region-specific antisera would suggest a high degree of primary structural conservation within the gastropod molluscs, but lack of conservation of the N-terminal region of the peptide in other invertebrate groups.     

Antisera raised against eledoisin and kassinin detect immunoreactive material in rat tissue extracts: tissue distribution and chromatographic characterization

Radioimmunoassays were developed for the tachykinins eledoisin (ELE) and kassinin (KAS) using antisera raised in rabbits. The antisera exhibited low (less than 0.1%) cross-reactivities to substance P (SP) and physalaemin (PHY), but crossreacted (with one exception, antiserum K7) to varying extents with neurokinin A (NKA) and neurokinin B (NKB). In the rat, the tissue distribution of the immunoreactive material detected by antiserum (E7) raised against ELE and by another antiserum (K1) raised against KAS both resembled that previously described for SP. Using the highly KAS-specific antiserum K7, no or only very low levels of immunoreactivity could be detected in extracts of various rat tissues. Gel permeation chromatography and ion-exchange chromatography of tissue extracts indicated that all antisera (except K7) detected the same population of immunoreactive molecules. One of the components was chromatographically indistinguishable from NKA. The tissue distribution of this component also resembled that of SP. Another immunoreactive component co-chromatographed with NKB at cation exchange chromatography. Acid tissue extracts, but not neutral tissue extracts, were found to contain immunoreactive components which appeared more basic than NKA and NKB. The total levels of immunoreactivity were higher in neutral than in acid tissue extracts. However, the ratio between the amounts of immunoreactivities in the two types of extracts varied considerably between tissues, indicating that tachykinin immunoreactive components may be present in different relative proportions in various tissues.     

Multiple tachykinins (neurokinin A, neuropeptide K and substance P) in capsaicin-sensitive sensory neurons in the guinea-pig

The occurrence of tachykinins in sensory neurons of the guinea-pig was studied by means of radioimmunoassay combined with ion-exchange and high-performance liquid chromatography as well as by immunohistochemistry. Antisera raised against kassinin (antiserum K12), neurokinin A (NKA) (antiserum NKA2) and substance P (SP) (antisera SP25 and SP2) were used. Antiserum K12 detected NKA, neuropeptide K (NPK) and a component eluting in the position of eledoisin (ELE) in extracts of the lung and ureter. Neurokinin B (NKB) was, however, not found. Neutral water extraction favored recovery of NKA and of the ELE-like component, while NPK was found only in acid extracts. The SP antisera detected two immunoreactive components of which the major form coeluted with synthetic SP. Capsaicin pretreatment depleted all these various forms of immunoreactivity in several peripheral organs including the ureter and lung. The immunoreactivity detected by antisera K12 or SP25 in radioimmunoassay had a similar regional distribution pattern in peripheral tissues. Immunohistochemical examination revealed that antiserum NKA2 stained the same spinal ganglion cells as the SP2 antiserum. The distribution of capsaicin-sensitive nerve fibers stained by these two antisera was also identical in peripheral organs such as the ureter, inferior mesenteric ganglion, heart and lung. It is concluded that multiple tachykinins, including SP, NKA, NPK and an ELE-like peptide, are present in capsaicin-sensitive sensory nerves in the guinea-pig. This finding can most likely be related to the origin of SP, NKA and NPK from the same precursor molecule, subsequent posttranslational tissue processing and axonal transport to terminal regions.     

Antisera raised against eledoisin and kassinin detect elevated levels of immunoreactive material in plasma and tumor tissues from patients with carcinoid tumors

Radioimmunoassays based on antisera raised against the tachykinins eledoisin (antiserum E7) and kassinin (antiserum K12) were used to measure the concentration of tachykinin-like immunoreactivity (TKLI) in plasma from 52 healthy subjects. 65 patients with carcinoid tumors (of which 46 had symptoms of both flushing and diarrhoea), and 6 patients with endocrine pancreatic tumors. The antisera did not crossreact with substance P (SP). Elevated concentrations of TKLI, as compared with healthy subjects, were found in 75% of the carcinoid patients, but in none of the patients with pancreatic tumors. Tumor metastases from 8 of the carcinoid patients all contained TKLI. Ion-exchange chromatography of plasma samples and tumor tissue extracts indicated the presence of several immunoreactive molecular forms. The elution patterns of the immunoreactivity detected by antisera E7 and K12 were similar, indicating that the same molecular species are measured by these antisera. None of the components coeluted with synthetic SP. One of the immunoreactive components in carcinoid tumor extracts coeluted with synthetic NKA. The major immunoreactive components in plasma from the patients eluted in a position different from that of all currently known mammalian tachykinins. Tachykinin immunoreactive material detected in tumor tissue and plasma of patients with carcinoid tumor may play a role in the symptomatology of the carcinoid syndrome.     

Tachykinin immunoreactivity in the european common frog,Rana temporaria: Localization,quantification and chromatographic characterization

1. Tachykinin immunoreactivity has been localized, quantified and chromatographicallycharacterized in the brain, stomach, intestine and skin of Rana temporaria.2. Antisera to mammalian substance P (SP) and neurokinin A (NKA) immunostained nerve fibres in all tissues except skin, and a population of mucosal endocrine cells in the intestinal epithelium.3. Radioimmunoassay of tissue extracts identified SP immunoreactivity in all tissues but NKA immunoreactivity was restricted to the brain.4. Chromatographie analysis of both frog tachykinins revealed that they possessed different physicochemical properties than their mammalian counterparts.     

The distribution and origin of nerve fibers immunoreactive for substance P and neurokinin A in the anterior buccal gland of the rat

The distribution and origin of substance P (SP) and neurokinin A (NKA) were studied in rat in the anterior buccal glands, which are minor mucous salivary glands. Indirect immunofluorescence staining showed moderate SP and NKA innervation of salivary acini and interlobular ducts, whereas blood vessels were more sparsely innervated, and there were few nerve fibers in the stroma and around the intralobular ducts. About 10%–20% of the trigeminal ganglion cells showed equally strong immunoreactivity to both SP and NKA. Unilateral denervation of the branches of the trigeminal nerve caused complete disappearance of the stromal fibers and greatly reduced the number of all other SP-immunoreactive and NKA-immunoreactive nerve fibers. In the superior cervical ganglia, SP and NKA immunoreactivity was restricted to small intensely fluorescent cells; SP and NKA immunoreactivity was absent from principal ganglionic cells, and thus sympathectomy had no any effect on the number or distribution of fibers immunoreactive for SP and NKA in the anterior buccal glands. The fibers remaining after sensory denervation could have been of parasympathetic origin, indicating a dual origin of nerves immunoreactive for SP and NKA in these glands. The present data demonstrate that the major part of the glandular SP and NKA innervation in the minor salivary glands derives from the trigeminal ganglia. The distribution of the peripheral nerve fibers indicates that they may play a role in the delivery of potent neuropeptides involved in the vascular, secretory, and motor (myoepithelial cells) functions of salivary glands.     

Tachykinin multiplicity in rat central nervous system as studied using antisera raised against substance P and neurokinin A

Antisera were raised in rabbits against the tachykinins neurokinin A (NKA) and substance P (SP). All NKA-antisera tested cross-reacted markedly with NKB, kassinin and eledoisin in radioimmunoassay (RIA), but virtually not with SP and physalaemin. Also when used for immunohistochemistry, one of the NKA-antisera was found to be virtually without cross-reactivity with SP. The most specific SP-antiserum did not cross-react with NKA but to some extent with NKB at the immunohistochemical level. Using these two antisera, the same distribution pattern of immunoreactivity was seen in both the rat substantia nigra and dorsal spinal cord. In neutral extracts of the substantia nigra, all NKA-antisera used for RIA detected a major component which eluted at the position of NKA in reverse phase high performance liquid chromatography, while no or only little immunoreactivity was detected at the position of NKB. A major component of substance P-like immunoreactivity (SPLI) co-eluting with SP and one or two minor SPLI-components were also detected in these extracts. An SP-antiserum, which cross-reacted markedly with physalaemin, detected an additional rather prominent component. In neutral water extracts of dorsal spinal cord the component detected with the NKA-antisera at the position of NKB, as well as one of the SPLI-components not eluting in the position of SP, were much more prominent than in the corresponding extracts of substantia nigra. In acetic acid extracts of both tissues, only one major SPLI-component co-eluting with SP could be detected, while only very small amounts of immunoreactivity eluting at the position of NKA and NKB (dorsal spinal cord only) could be detected using the NKA-antisera. The present results illustrate the importance of the extraction method used in immunochemical studies and demonstrate that the relative proportions of various tachykinins are markedly different in the rat substantia nigra and dorsal spinal cord.     

The primary structure of peptide Y (PY) of the spiny dogfish, Squalus acanthias: immunocytochemical localisation and isolation from the pancreas.

1. Endocrine cells within islets, exocrine parenchyma and ductal epithelium in the pancreas of the spiny dogfish, Squalus acanthias, were immunostained with an antiserum to the C-terminal region of mammalian neuropeptide Y (NPY). 2. Radioimmunoassay of pancreatic extracts with the same antiserum detected immunoreactivity in the dorsal lobe (338 pmol/g) and ventral lobe (433 pmol/g). Reverse phase HPLC analysis of both extracts resolved a single immunoreactive peptide. 3. The primary structure of the isolated peptide was established as: YPPKPENPGEDAPPEELAKYYSALRHYINLITRQRY.NH2. 4. Peptide Y (PY) from Squalus acanthias is identical in primary structure to an NPY-related peptide isolated from the pancreas of Scyliorhinus canicula and has a 31/36 residue homology with porcine NPY. The 5 substitutions are highly-conservative.     

Tachykinin immunoreactivity in the European common frog, Rana temporaria: localization, quantification and chromatographic characterization.

1. Tachykinin immunoreactivity has been localized, quantified and chromatographically-characterized in the brain, stomach, intestine and skin of Rana temporaria. 2. Antisera to mammalian substance P (SP) and neurokinin A (NKA) immunostained nerve fibres in all tissues except skin, and a population of mucosal endocrine cells in the intestinal epithelium. 3. Radioimmunoassay of tissue extracts identified SP immunoreactivity in all tissues but NKA immunoreactivity was restricted to the brain. 4. Chromatographic analysis of both frog tachykinins revealed that they possessed different physico-chemical properties than their mammalian counterparts.     

A novel radioimmunoassay for neuromedin K. I. Absence of neuromedin K-like immunoreactivity in guinea pig ileum and urinary bladder. II. Heterogeneity of tachykinins in guinea pig tissues

A novel and highly specific radioimmunoassay for the tachykinin peptide neuromedin K (NMK, also known as neurokinin beta, neurokinin B) has been developed and used to determine the distribution of this peptide in extracts of guinea pig tissues. In addition to immunoreactive components coeluting with the 3 mammalian tachykinins, substance P (SP), substance K (SK) and NMK, analyses using reverse-phase HPLC revealed immunoreactive peaks coeluting with the C-terminal octapeptide of SK (SK-(3-10], an N-terminally extended form of SK (gamma-preprotachykinin-(72-92)amide), and a yet unidentified peak eluting before NMK in the extracts of guinea pig brain and spinal cord. In contrast to the other tachykinins, SP and SK, which were present in high concentrations in extracts of all peripheral and central tissues examined, NMK-like immunoreactivity was detected only in extracts of central tissues. NMK-like immunoreactivity was not detected in extracts of terminal ileum and urinary bladder.     

Localization of the myomodulin-like immunoreactivity in the leech CNS

The distribution of myomodulinlike immunoreactivity in the leech CNS was determined using an antiserum raised against Aplysia myomodulin. Segmental ganglia contained approximately 60 immunoreactive neurons. In addition, numerous fibers containing immunoreactive varicosities were found throughout the neuropil. Using a combination of Lucifer Yellow injections and immunocytochemistry, we identified neurons including the anterior Pagodas (AP), annulus erector (AE), motor neurons, Leydig, longitudinal muscle motoneurons (L), S cells, and coupling interneurons, all of which are active during the touch-elicited shortening reflex. FMRF-amide-like immunoreactivity in three of these cells (L, AP, and AE) was previously demonstrated. Specific staining for myomodulin was abolished by preadsorption of the antiserum with synthetic myomodulin, but not with FMRF-amide. These results suggest a potential role for myomodulin in both intrinsic and extrinsic modulation of the leech touch-elicited shortening reflex. Further, it is possible that several neurons mediating this reflex contain multiple neuromodulatory peptides. © 1996 John Wiley & Sons, Inc.     

FMRFamide immunoreactive neurons in the central nervous system of the snail,Achatin a fulica

• 1.1. FMRFamide immunoreactive neurons were detected in the central nervous system of the snail, Achatina fulica.
• 2.2. FMRFamide immunoreactive neurons were found in all the ganglia comprising the central nervous system. In particular, the immunoreactivity was recognized in both the ordinary and giant neurons of the visceral and right parietal ganglia.
• 3.3. In the cerebral and pleural ganglia, FMRFamide immunoreactive neurons were found only in the ordinary neurons. The immunoreactivity was shown to have a tendency to form a group in the cerebral and pedal ganglia.

     

An FMRFamide antiserum differentiates between populations of antigens in the ventral nervous system of the locust,Schistocerca gregaria

Summary The distribution of FMRFamide immunoreactive neurones in the ventral nerve cord of the locust, Schistocerca gregaria, is described. These neurones are found only in the suboesophagael and thoracic ganglia, although immunoreactive processes are found in the neuropils of the abdominal ganglia. Many of these neurones also react with an antiserum raised against bovine pancreatic polypeptide (BPP), but this antiserum also reveals another population of cells in the abdominal ganglia. The staining obtained with the BPP antiserum is blocked by preabsorption of the antiserum with FMRFamide; the converse is not true: FMRFamide-like immunoreactivity is not suppressed by preincubation with BPP. These results suggest that there are at least two endogenous peptide antigens in the locust nerve cord: one is found in cells of the suboesophageal and thoracic ganglia, and the other is found in cells of the abdominal ganglia.     

Occurrence and effects of multiple tachykinins; substance P, neurokinin A and neuropeptide K in human lower airways

In the present work we have studied the occurrence of different tachykinins (substance P (SP), neurokinin A (NKA) and neuropeptide K (NPK)) in human distal bronchi and pulmonary arteries by means of radioimmunoassay (RIA) and high performance liquid chromatography (HPLC). We have also compared the biological effects of different tachykinins on isolated human bronchi and pulmonary arteries in vitro. The concentration of immunoreactive SP using antiserum SP2 in the pulmonary arteries was higher (1.34 +/- 0.15 pmol/g) than in the bronchi (0.56 +/- 0.05 pmol/g). The contents of other tachykinins than SP measured using antiserum K12 was on the other hand considerably higher in the bronchi (0.33 +/- 0.14 pmol/g) than in pulmonary arteries (0.13 +/- 0.02 pmol/g). Immunoreactive materials corresponding to SP, NKA and NPK were identified in bronchial extracts by RIA combined with HPLC, which also indicated the presence of an eledoisin (ELE)-like component. In vitro studies showed that NKA was the most potent of the tachykinins as a bronchoconstrictor agent, being several hundred-fold more active than SP, acetylcholine and histamine. NPK had an intermediate potency. The bronchoconstrictor effect of NKA was unaffected by atropine, mepyramine and cimetidine. The tachykinins SP and NKA had on the other hand, a rather equal potency in inducing relaxation of serotonin precontracted pulmonary arteries. In conclusion, multiple tachykinins are present in lower airways of man. These peptides exert different biological activities whereby NKA is a very active bronchoconstrictor agent compared to SP while both NKA and SP have rather similar relaxatory activities of vascular smooth muscle.     

Sensory innervation of the ear drum and middle-ear mucosa: Retrograde tracing and immunocytochemistry

Summary The distribution and origin of nerve fibers of presumed sensory nature in the ear drum and middle-ear mucosa of the rat were studied by a retrograde tracing technique in combination with immunocytochemistry.Application of True Blue (TB) on the ear drum or on the middle-ear mucosa labeled nerve cell bodies in the jugular, trigeminal, geniculate and cervical dorsal root ganglia (C₂–C₄). Judging from the number of TB-labeled nerve cell bodies the jugular and trigeminal ganglia contributed the major component to the sensory innervation of the ear drum and the middle-ear mucosa, while the contribution from the geniculate and cervical dorsal root ganglia was relatively minor.The majority of the TB-labeled nerve cell bodies contained calcitonin gene-related peptide (CGRP), whereas minor populations stored substance P (SP) and neurokinin A (NKA). Nerve fibers containing SP, NKA and CGRP were moderate in number in the middle-ear mucosa and few in the ear drum. Double immunostaining revealed that SP invariably coexisted with NKA in nerve cell bodies in the ganglia examined. The SP/NKA-containing nerve cell bodies constituted a subpopulation of those storing CGRP.The findings indicate that several ganglia project to the ear drum and middle-ear mucosa and that many neuropeptides are involved in the mediation of middle-ear sensitivity.     

Development of substance P and neurokinin A immunoreactivity in ganglia supplying nerves to the submandibular glands of the rat

Developing submandibular, trigeminal and superior cervical ganglia, which provide innervation to the submandibular glands, were studied for substance P (SP)-and neurokinin A (NKA)-immunoreactive (IR) ganglion cells and nerve fibres in rat. These ganglia were examined by using an indirect immunofluorescence technique at daily intervals from the 16th day in utero (i.u.) until birth, and subsequently on the 2nd, 5th, 7th, 12th, 16th, 30th, 42nd postnatal day and in the adult (3 months). In the submandibular ganglion SP- and NKA-IR cells and fibres first appeared in considerable numbers on the 19th day i.u. (in one sample out of five on the 18th day i.u.), when more than 90% of the ganglion cells were immunoreactive to SP and NKA. The number stayed at more than 90% to the 7th postnatal day and then slowly decreased to the levels of adult animals (18% SP, 17% NKA). The first SP- and NKA-IR ganglion cells and fibres appeared in the trigeminal ganglion on the 18th day i.u. when they represented 7% (SP) and 4% (NKA) of the ganglion cells. The number of SP- and NKA-IR cells increased steadily, reaching a maximum at the time of birth when 68% (SP) and 74% (NKA) of the ganglion cells were immunoreactive. Thereafter they began to decrease toward the level of an adult rat (10% SP, 11% NKA). In the superior cervical ganglion only a few SP-and NKA-IR ganglion cells were detected from the 19th day i.u. to the fifth postnatal day. Positive ganglion cells were also occasionally found in the nerve trunks outside the superior cervical ganglion. From the seventh day onwards no SP- or NKA-IR ganglion cells were found. SP-and NKA-IR SIF (small intensively fluorescent) cells were detected from the 16th postnatal day onwards.     

Distribution and partial characterization of CREB-like immunoreactivity in the medicinal leech Hirudo

The presence and distribution of immunoreactivity to the cyclic AMP response element binding protein (CREB) were determined in the central nervous system (CNS) and in peripheral tissues of the medicinal leech Hirudo. Western blots revealed several CREB-immunoreactive (CREB-IR) bands including one whose molecular weight (43–44 kDa) was similar to mammalian CREB. The 43–44 kDa CREB-like protein was detected in nuclear extracts of the ventral nerve cord and was not observed following preincubation of the primary antiserum with the epitope sequence. CREB-like immunoreactivity was detected in extracts from each of six regions of the leech CNS, and in extracts from leech body wall musculature, crop, intestine, jaw musculature, pharynx, and salivary tissues. Whole mounts of leech ganglia revealed specific CREB-IR in a restricted population of neurons distributed throughout the leech CNS. Apparent homologues to a pair of CREB-IR dorsolateral neurons were observed in most ganglia along the ventral nerve cord. Several CREB-IR neurons exhibited segmental specificity. A number of neurons stained with an antiserum to the cyclic AMP response element modulator (CREM). These neurons showed no overlap in location with CREB-IR neurons, and this staining was not eliminated with a preabsorption control. Possible roles for a CREB-like protein in the leech are discussed. Electronic Publication     

The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa

Enzyme histochemistry and immunocytochemistry were used to determine the distribution of neurons in the snail Helix aspersa which exhibited nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and/or immunoreactivity to nitric oxide synthase (NOS). NADPH diaphorase-positive cells and fibres were distributed extensively throughout the central and peripheral nervous system. NADPH diaphorase-positive fibres were present in all neuropil regions of the central and peripheral ganglia, in the major interganglionic connectives and in peripheral nerve roots. NADPH diaphorase-positive cell bodies were found consistently in the eyes, the lips, the tentacular ganglia and the procerebral lobes of the cerebral ganglia; staining of cell bodies elsewhere in the nervous system was capricious. The distribution of NOS-like immunoreactivity differed markedly from that of NADPH diaphorase activity. Small clusters of cells which exhibited NOS-like immunoreactivity were present in the cerebral and pedal ganglia; fibres which exhibited NOS-like immunoreactivity were present in restricted regions of the neuropil of the central ganglia. The disjunct distributions of NADPH diaphorase activity and NOS-like immunoreactivity in the neurvous system of Helix suggest that the properties of neuronal NOS in molluscs may differ sigificantly from those described previously for vertebrate animals.     

Small cardioactive peptide-like immunoreactivity and its colocalization with FMRFamide-like immunoreactivity in the central nervous system of the leech Hirudo medicinalis

Summary The distributions of small cardioactive peptide (SCP)- and FMRFamide-like immunoreactivities in the central nervous system of the medicinal leech Hirudo medicinalis were studied. A subset of neurons in the segmental ganglia and brains was immunoreactive to an antibody directed against SCP_B. Immunoreactive cell bodies were regionally distributed throughout the nerve cord, and occurred both as bilaterally paired and unpaired neurons. The majority of the unpaired cells displayed a tendency to alternate from side to side in adjacent ganglia. A small number of neurons were immunoreactive only in a minority of nerve cords investigated. Intracellular injections of Lucifer yellow dye and subsequent processing for immunocytochemistry revealed SCP-like immunoreactivity in heart modulatory neurons but not in heart motor neurons. FMRFamide-like immunoreactivity was also detected in cell bodies throughout the central nervous system. A subset of neurons contained both SCP- and FMRFamide-like immunoreactivities; others stained for only one or the other antigen. These data suggest that an antigen distinct from FMRFamide is responsible for at least part of the SCP-like immunoreactivity. This antigen likely bears some homology to the carboxyl terminal of SCP_A and SCP_B.     

Regional distribution and in vivo release of tachykinin-like immunoreactivities in rat brain: evidence for regional differences in relative proportions of tachykinins

The regional distribution of various forms of tachykinin-like immunoreactivity (TKLI) was studied in rat brain using radioimmunoassay. TKLI was measured with two different tachykinin-antisera (K12 and E7), which react with neurokinin A (NKA) and neurokinin B (NKB) but not with substance P (SP) and with a specific SP-antiserum. TKLI-K12 and TKLI-E7 were found to have similar regional distributions which were, however, significantly different from that of the substance P-like immunoreactivity (SPLI). Thus, the ratio of the tissue concentrations of TKLI-K12 or TKLI-E7 to that of SPLI was higher in frontal cortex and hippocampus and lower in pons/medulla oblongata than in the other regions studied. Cation-exchange chromatography of neutral water extracts of brain tissue revealed two major immunoreactive components of TKLI-K12 and TKLI-E7, one of which co-eluted with synthetic NKB while the other appeared in the same region as synthetic NKA. The relative quantities of these components varied depending on the brain region studied. No TKLI-K12 or TKLI-E7 co-eluted with synthetic SP. Almost all of the SPLI in acetic acid or water extracts of brain tissue eluted as a single chromatographic component in the same position as synthetic SP. Potassium-stimulated in vivo release of TKLI-K12, TKLI-E7 and SPLI in striatum of rat brain could be demonstrated using intracerebral dialysis. The present results imply that tachykinins, which may serve as neurotransmitters or neuromodulators, are present in different proportions in different regions of rat brain.