Tomas Hökfelt

Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons.

With the indirect immunofluorescence technique of Coons and collaborators the occurrence of substance P (SP)-like immunoreactivity was studied in spinal ganglia (L6-S1), the spinal cord (L6-S1) and the pad skin of the hind paw of the cat. In untreated cats a very dense network of SP-positive fibers was found in the spinal cord in Lissauers fasciculus, in laminae I-III and a rather dense plexus was seen in the ventral horns, in the area around the central canal (laminae X) and in the medial parts of laminae VI and VII. SP-positive fibers were also observed in the connective tissue under the epithelium of the skin. However, in untreated cats no specific immunogluorescnece was observed in the spinal ganglia, dorsal roots or certain large peripheral nerve trunks. After certain experimental procedures such as local application of colchicine or compression of the dorsal root close to the spinal ganglion, SP-positive fluorescence was observed in a rather small number of neuronal cell bodies and in fibers. The fluorescent material was observed in the peripheral parts of the cytoplasm and the cell bodies were exclusively of the small type. Ten days after transection of the dorsal roots a marked decrease in the number of SP-positive fibers was observed in the substantia gelatinosa but not in the ventral horns. The present results give strong evidence for the occurrence of SP in a certain population of primary sensory neurons and support earlier findings that SP may act as a transmitter or modulator in these neurons.

Evidence for dopamine receptor stimulation by apomorphine

Sm,-Recently, Ernst (1967) has reported that the apomorphine-induced compulsive gnawing in rats is not mediated via the release of catecholamines, since it is not reduced by the catecholamine synthesis inhibitors a-methyl-3,4dihydroxyphenylalanine and a-methyltyrosine. On the other hand, the gnawing seen after treatment with (+)-amphetamine is blocked by these synthesis inhibitors. Since the apomorphine-induced gnawing requires an intact corpus striatum and gnawing can also be produced by the catecholamine precursor dihydroxyphenylalanine, Ernst (1967) suggested that apomorphine acts on the dopamine receptors whereas amphetamine acts by releasing dopamine. In the present paper supporting evidence for this view is given by further functional, biochemical and histochemical studies. The functional influence of apomorphine on dopamine neurotransmission in the corpus striatum was examined after unilateral removal of the corpus striatum of adult hooded rats weighing about 200 g (And& Dahlstrom & others, 1966a). A possible action of apomorphine on the noradrenaline receptors of the spinal cord was tested in acutely spinalized adult hooded rats by evaluating the changes in the flexor reflex evoked by pinching the hind limbs. The effect of apomorphine on the dopamine and noradrenaline levels of the brain and spinal cord was determined biochemically (Bertler, Carlsson & Rosengren, 1958; Carlsson & Waldeck, 1958) and histochemically (Falck, Hillarp & others, 1962; Dahlstrom & Fuxe, 1964; Hamberger, Malmfors & Sachs, 1965). Function. These studies were made mainly on rats which had been pretreated with reserpine (10 mg/kg i.p., 3 hr) plus a-methyltyrosine methylester (H 44/68, 500mg/kg, i.p., 2 hr) after removal of the left corpus striatum by suction. After this treatment all the operated animals turned towards the unoperated side (cf. And6n & others, 1966a). After injection of apomorphine (1-25 mg/kg s.c.) these rats changed their position and turned or rotated towards the operated side. This effect began about 5 min after the injection and was evident for about 45-60 min. If apomorphine was given to operated rats not pretreated with reserpineH 44/68 combination, this action of apomorphine, like the gnawing, seemed to be less pronounced. If haloperidol (5 mg/kg i.p.) was given 15-20 min after apomorphine all the rats turned from the operated towards the unoperated side in about 15 min and the gnawing ceased. (+)-Amphetamine (0.5-25 mg/kg s.c.), like apomorphine, made the rats turn or rotate towards the operated side. In contrast to apomorphine, however, this action of amphetamine was not seen after pretreatment with reserpine plus H 44/68 (cf. Weissman, Koe & Tenen, 1966; Hanson, 1967; Ernst, 1967). Apomorphine (25 mg/kg s.c.), in contrast to (+)-amphetamine (05-25 mg/kg s.c.) and ~-3,4-dihydroxyphenylalanine (50-75 mg/kg i.v. 2 hr after nialamide 50 mg/kg i.p.), did not cause a definite increase of the flexor reflex in spinalized rats. Chemistry. The biochemical results obtained in unoperated adult hooded rats are presented in Table 1. Apomorphine caused a retardation of the depletion in brain dopamine produced by H 44/68. The difference between the dopamine levels in the apomorphine-H 44/68 group and in the H 44/68 group is statistically significant (P < 0.001, Student’s r-test). This action of apomorphine on the brain dopamine was blocked by haloperidol. The disappearance of noradrenaline from the brain and the spinal cord after H 44/68 did not seem to be influenced by apomorphine. (+)-Amphetamine did not cause any significant retardation of the dopamine and noradrenaline loss after H 44/68.

Immunohistochemical evidence of substance P-like immunoreactivity in some 5-hydroxytryptamine-containing neurons in the rat central nervous system.

Abstract With the indirect immunofluorescence technique of Coons and collaborators a possible coexistence of 5-hydroxytryptamine (5-HT) and substance P in neurons of the lower medulla oblongata was explored. Antisera to 5-HT and to dopadecarboxylase (aromatic l -aminoacid decarboxylase), an enzyme probably present in immunologically indistinguishable forms both in catecholamine and 5-HT neurons, were used as markers for 5-HT neurons and an antiserum raised to synthetic substance P conjugated with bovine serum albumin for substance P-containing neurons. Five or 10 μm thick, consecutive sections were stained with the three antisera. Numerous cell somata in nucleus raphe magnus, nucleus raphe obscurus, nucleus raphe pallidus, pars α of the nucleus reticularis gigantocellularis and nucleus interfascicularis hypoglossi contained both substance P-like immunoreactivity and 5-HT (and dopadecarboxylase) immunoreactive material. After intraventricular or intracisternal injections of 5,6- or 5,7-dihydroxytryptamine, two neurotoxins assumed to cause degeneration mainly of 5-HT neurons, enlarged substance P and 5-HT (and dopadecarboxylase) positive fibres were seen in, around and lateral to the olivary complex. Furthermore, in these rats both substance P and 5-HT positive nerve terminals in the ventral horns of the spinal cord disappeared. These findings indicate that substance P and 5-HT may coexist not only in some cell bodies but also in axons and nerve endings. The latter conclusion must, however, remain tentative since the neurotoxins may cause unspecific damage and thus not only damage 5-HT (and postulated ‘SP-5-HT’) neurons. In further experiments reserpine was used, a drug known to deplete monoamines by affecting their storage sites. With a high dose of reserpine a marked depletion of 5-HT was obtained both in nerve terminals and cell bodies whereas substance P immunoreactive material seemed unaffected. Possible interpretations of these findings is that substance P and 5-HT have different storage sites within the neuron, or that reserpine selectively causes loss of 5-HT and not substance P from the same storage site.

Messenger plasticity in primary sensory neurons following axotomy and its functional implications

Following peripheral axotomy, long-lasting changes in the expression of neuropeptides and their receptors in primary sensory neurons are observed. These changes involve the downregulation of the excitatory peptides substance P and calcitonin gene-related peptide and the upregulation of the inhibitory peptides neuropeptide tyrosine and galanin, resulting in a reduction of transmission in the dorsal horn. The changes observed are thought to represent adaptive responses to limit the consequences of peripheral nerve damage to the organism as a whole and to promote survival and recovery of the individual neuron.

The distribution of enkephalin-immunoreactive cell bodies in the rat central nervous system ☆

With the indirect immunofluorescence technique the distribution of methionine-enkephalin-immunoreactive cell bodies was studied in the central nervous system of rats pretreated with colchicine. The antiserum used did cross-react to 10% with leucine-enkephalin but to less than 0.1% with alpha-, beta-, and gamma-endorphine. Cell bodies with a specific immunofluorescence were observed in the tel-, di-, mes- and rhombencephalon and in the spinal cord.

Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactive ganglion cells

SummaryBy use of the indirect immunofluorescence technique the distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) has been analyzed in cervical and lumbar dorsal root ganglia of untreated and colchicine-treated rats. In addition, lumbar ganglia were examined 2 weeks after transection of the sciatic nerve. The occurrence of CGRP-positive cells in relation to ganglion cells containing substance P-, somatostatin-, galanin-, cholecystokinin (CCK)-, and vasoactive intestinal polypeptide (VIP)/peptide histidine isoleucin (PHI)-LI has been evaluated on consecutive sections as well as using elution-restaining and double-staining techniques.CGRP-LI was observed in many ganglion cells of all sizes ranging in diameter from 15 μm to 65 μm. Thus, this peptide occurs also in the large primary sensory neurons. In contrast to the sensory peptides described to date, CGRP-positive cells constituted up to 50% of all and 70% of the medium-sized neurons, thus being the most frequently occurring peptide in sensory neurons so far encountered. Subpulations of CGRP-positive neurons were shown to contain substance P-, somatostatin-, or galanin-LI and some CGRP-positive neurons contained both substance P- and galanin-LI. In fact, most substance P-, somatostatin- and galanin-positive cell bodies were CGRP-immunoreactive. The coexistence analysis further revealed that galanin and substance P often coexisted and that some cells contained both substance P- and somatostatin-LI, whereas no coexistence between galanin and somatostatin has as yet been seen. VIP/PHI-LI was only shown in a few cells in untreated or colchicine-treated rats. However, after transcetion of the sciatic nerve numerous VIP/PHI-positive cells were observed, some of which also contained CGRP-LI.The present results indicate that a CGRP-like peptide is present in a wide range of primary sensory neurons probably not related to specific sensory modalities. Often this peptide coexists with other biologically active peptides. Taken together these findings suggest that CGRP may have a generalized function.

Hypocretin/Orexin- and melanin-concentrating hormone-expressing cells form distinct populations in the rodent lateral hypothalamus: Relationship to the neuropeptide Y and agouti gene-related protein systems

Cells in the lateral hypothalamus and in the arcuate nucleus play prominent roles in the central control of food intake; however, a neurochemical link connecting these potential components of a hypothalamic circuitry regulating energy metabolism remains to be established. In the present study, the topographical relationship between cells expressing mRNAs encoding melanin‐concentrating hormone and the newly discovered neuropeptide family hypocretins/orexins was studied in the rat and mouse lateral hypothalamus by using double‐labeling in situ hybridization. Cells expressing the two mRNAs formed completely distinct populations, with hypocretin/orexin cells located primarily perifornically and in the magnocellular lateral hypothalamic nucleus; melanin‐concentrating hormone cells extended in a wider area both laterally and periventricularly and appeared to partly surround the hypocretin/orexin population. In the arcuate nucleus, cells expressing neuropeptide Y and agouti gene‐related protein were studied by routine fluorescence and/or confocal microscopy immunohistochemistry. Double staining demonstrated that a large proportion of the neuropeptide Y‐positive cell bodies in this nucleus also contained agouti gene‐related protein‐like immunoreactivity. Moreover, these two peptides also coexisted in nerve terminals surrounding and in close relationship to perikarya and processes of both hypocretin/orexin‐ and melanin‐concentrating hormone‐immunoreactive cells in the lateral hypothalamus, whereby the former appeared to receive a more dense innervation. These results thus provide evidence for an arcuate‐lateral hypothalamic neuropeptide Y/agouti gene‐related protein pathway. Furthermore, the results implicate hypocretin/orexin and melanin‐concentrating hormone‐expressing cells as downstream targets in neuropeptide Y‐induced feeding. J. Comp. Neurol. 402:460–474, 1998.

Effect of antidepressant drugs on the depletion of intraneuronal brain 5-hydroxytryptamine stores caused by 4-methyl-α-ethyl-meta-tyramine

Abstract Mice were injected with 4-methyl-α-ethyl-meta-tyramine (two doses of 100 mg/kg i.p.). After 4 h the animals were killed and the brains analyzed for 5-hydroxytryptamine (5-HT) biochemically or examined under the fluorescence microscope after formaldehyde treatment. The amine caused more than 50 per cent depletion of 5-HT, probably by displacement. Pretreatment with antidepressant drugs of imipramine type largely prevented 5-HT depletion, probably by blocking the amine uptake mechanism of the cell membrane of 5-HT neurons. Tertiary amines, e.g. chlorimipramine, imipramine and amitriptyline, were more potent in this respect than the corresponding secondary amines, in contrast to previous observations on noradrenaline neurons. In rats similar observations were made (histochemical observations only). It is suggested that blockade of 5-HT re-uptake is involved in the mood-elevating action of tricyclic antidepressants, whereas blockade of noradrenaline re-uptake promotes drive in the depressed patient.

Co-existence of substance P and calcitonin gene-related peptide-like immunoreactivities in sensory nerves in relation to cardiovascular and bronchoconstrictor effects of capsaicin.

Immunohistochemical studies showed that substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactivity co-exist in capsaicin-sensitive primary sensory neurons. Varicose SP- and CGRP-immunoreactive nerve fibres with a similar distribution pattern were seen in the lower airways and heart. The functional analysis revealed that CGRP caused cardiac stimulation and had, together with SP and neurokinin A, potent hypotensive effects. Vascular permeability was increased by SP and neurokinin A, and the bronchial smooth muscle was particularly sensitive to neurokinin A. Thus, multiple peptides stored in an possible released from the same nerve endings by capsaicin may exert differential effects in various target tissues.

Evidence for a central noradrenaline receptor stimulation by clonidine

Abstract Clonidine increased the flexor reflex of spinal rats also after depletion of all known noradrenaline stores, indicating a stimulation of also central noradrenaline receptors. No stimulation of 5-hydroxytryptamine or dopamine receptors was observed. The motility of reserpine treated animals was enhanced by clonidine provided that the DA receptors were stimulated. Clonidine reduced the disappearance of noradrenaline and also that of 5-hydroxytryptamine after synthesis inhibition. The chemical effects might be due to a negative feedback mechanism, evoked by the noradrenaline receptor stimulation. Both the functional and chemical changes were reduced by the noradrenaline receptor blocking agents haloperidol and phenoxybenzmine.