Histamine as a transmitter in brain

Life Sciences Vol . 17, pp . 503-518 Printed in the II .S .A .

Pergamoa Press

MINIREVISW SISTAI~iB AS A TRANSI~TTSR IDi BBAIN Jean-Charles Schwartz Unité de Neurobiologie (U .109) de l'Institut National de la Santé et de la Recherche Médicale ; 2 ter, rue d'Alésia ^ 75014 Paria - France . Histamine ie probably a neurotransmitter in mammalian brain. It is synthesised by a specific decarbozylase localized is the cytoplasm of aerve-endings, partly stored in synaptic vesicles, and during depolarization it is released and its synthesis accelerated . Specific receptors to this amine as evidenced by electrophysiological and behavioural studies, ae well as by the activation of cyclic AMP formation, are present in brain. Lesion studies indicate that histamine-containing neuroaea constitute as ascending bundle arising from the brain-stea, passing through the lateral hypothalamus, and projecting diffusely into the whole teleacephalon . This disposition, together with neuropharmacological data, suggest that hiataminargic neurones might be involved, like the monoaminergic ones, in the control of states of wakefulness and sleep . Additionally, ae indicated by histological and biochemical studies, a fraction of cerabial histamine ie held in meet-cells and, when released from the letters, might also play a "transmitter" role in immune or inflammatory processes . In one of the moat popular Handbooks of Pharmacology (1), the imidasolethylamiae, histamine (HA) is categorised in the class of "autacoida", i.e . substances which poeessa intense activity on a variety of biological systems, which are normally present is significant amounts in tiesues,~but whose physioFurthermore, the author of logical function is still a matter of controversy . the monograph uses a striking comparison, paraphrasing Pirandello, tells of Indeed, no clear pattern has yet emerged "Autacoids in search of a function". from the numerous studies attempting to uncover the possible biological signiNevertheless the situation ficance of this amine in peripheral tissues. appears to be somewhat better in the C.N .S ., and HA is now currently listed ae a putative neurotransmitter . That the cerebral amine could have this function was already suggested in 1970 by J.P . Green in his comprehensive review (2) but a large body of ea~perimental data has been accumulated in recent years which leads strong support to this idea . The present Minireview is concerned with those aspects of the question characterisation of the HA~ which have only recently found suitable answers : synthesizing enzyme, identification of the calls storing the amine, localiza tion of HA-containing neural pathways and characterization of the HA receptors in the mammalian brain . I : Biosynthesis of Histamine in Brain Hiatidine De~arbozylaee or Aromatic Aminoacid Decarbozylase ? This subject is not merely a question for biochemists since the possible esieteuce in brain of specific HA-containing neurones, distinct from the monoaminergic ones, depends on the answer . 503

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Ezperiments performed in several animal species have shown that labeled HA does not diffusa readilq from blood to brain (3, 4) indicating that the brain stores depend on local biosynthesis . HA formation is a simple, one-step process but tvo different enzymes from mamalian tissues are apparently able (5) to catalyse the decarborylation of the natural aminoacid, hiatidine : the aromatic-L-aminoacid decarbo~glase (EC 4 .1 .1 .26) and the "specific" L-hiatidine decarboz9lase (EC 4 .1 .1 .22) . If cerebral HA were synthesized by the former enzyme (found in high activity in catecholaminergic and serotoaiaergic neurones), this could rule out the presence of HA in a specific neuronal system . Obviously, a definitive aaewar to this critical problem should await the isolation in pure form of the cerebral enzyme decarbosylatiag L-hiatidine . There are, however, many other lines of evidence (kinetics, effects of inhibitors, regional distribution) to indicate that cerebral HA formation dapeada on a diatiact decarborylase (EC 4 .11 .22) . Ia homogenates of rat ]~ypotha lamua, the region with the highest activity, kinetic data for the HA-forming anzyioe (6) are very similar to those reported for the specific H.D . found for iastaace is the rat stomach i .e . the pH optimum of the enzyme is iav rsely related to the substrate concentration (with a value near t~ 7 .0 for 10~-hietidine) and its affinity ie relatively high (Rm near to 10 M) . On the other hand, the aromatic L-aminoacid decarbozylaee azhibite a much lower affinity for L-hietidine, or ie usable to decarborylate the latter (5) . When the above Sm value for H.D . is conaiderad, it wind appear tha~ the mean concentration for free L-hiatidine in brain tissues, i .e . around 10 M, is hardly sufficient to saturate the anayma . This situation resembles that of cen tral earotoniaergic neurones in which the enzyme tryptophaae hydrorylase is sot saturated by its substrate and suggests that the biosynthesis of HA might be physiologically influenced by fluctuations in the level of L-hiatidine in plasma and pharmacologically increased by loading the animals with the precursor aminoacid . This assumption appears to be confirmed by the effects of L-hiatidine loads administered to rats (7) or mice (8), since such treatment markedlq increased cerebral HA levels in regions (7) and eubcellular fractions (8) where the endogenous amine is normally concentrated . The differential affects of decarboaylase inhibitors, both in vitro and in viva , also indicate that a specific H .D . is responsible for cerebral HA biosyathe is . In hamoogenatea of rat hypothalamus, HA f rmstion was not affected by 10~ d-methyl-DOPA or partially inhibited by 10 ~ Ro 4-4602 although, at the same coaceatratioas, 0~-hydraziao-histidiae and brocraeine (NSD 1055), two known inhibitors of the specific H .D . blocked completely t1A formation (6) . Similar data have been recently provided for the HA-forming enzyme from brain tissues of mouse (8), guinea-pig (9), cat and even man (G . Barbin, M . Gar berg, J . Talairach and J .C . Schwartz, to be published) . The in viva affects of these inhibitors on 3HA formation in rat brain, evaluated after administration of a tracer dose of H-hiatidine or a loading dose of L-hiatidine, are also coasisteat with this view . Methyl-DOPA or Ro 4-4602, administered in doses high enough to prevent the increase of brain serotonin induced by 5-hydrozytryptophan, did not affect HA formation . Oa the other hand, administration of brocresina and K-hydrazine-hiatidine not only decreased markedly HA formation but induced a rapid (although partial) decline in the endogenous level of the cerebral amine (7, 8) . Further evidence for the postulate that the HA synthesizing enzyme in brain is diatiact from the non-specific aromatic-L-aminoacid decarbo :ylase arises from the comparison of the regional distributions of the tw types of enzyme activity . Despite some resemblances (high activities in the hypothala-

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mus, low activities in the cerebellum for both), the difference ie clear in the striatum, a region which holds only moderate H.D . activity (6) (see also table III),but contains th~ highest activity for the non-specific dacarbozylaea . The latter enzyme is present mainly is the dopaminergic neurones and, after degeneration of these neurones induced by 6-l~ydrozydopamiae, its activity decreases by about 50Z, while H.D . activity remains intact (11, 12) . Therefore, is vitro as well ae in viva data indicate that cerebral HA for motion depends upon a specific enzyme for which L-hietidine ie the only known substrate . Tha one possible ezceptioa ie the natural g+~~++~ acid tale N-msthyl hietidine from which tela N-methyl-histamine is fors~ed (13), a substance with little biologic active yand which ie also - and predominantly - produced is brain by tranemethylation of HA (14, 15) . II :

Histamine-Containing Cells In Brain :

Neurones or Mast-Calls ?

The uncovering of the physiological functions of cerebral HA requires the precise identification of the types of calls synthesizing and storing this amine . One major source of HA in peripheral tissues, including the sheaths of the peripheral nerves, is the mast-cell, a connective tissue call holding in its granules high amounts of heparin and HA (together with smaller amoimta of serotonin and dopamine, at least in some animal species) . The importance of the meet-cells as a potential store for HA in brain has been overloked for a long time because they have been only rarely encoumtered upon histological azamiaation of the mammalian brain (2) . Sowever, owing to the relatively low level of HA in brain (around 50 ng/g, i.e . about one tenth of the level of noradrenaline or serotonin), a relatively small number of mast-cp~lls containing HA is amounts similar to typical peritoneal maet-cells (13 ag/10 cells) could account for the whole amine content. In fact, several publications (16, 21) have recently reported the occureace of mast-cells, identified by classical staining or even by histofluorescance of HA (20, 21) in the mammalian brain. They were especially numerous in meniageB, along vessels but were also found in parenchyma . Although the comparison of brain regions based on precise and aztanaive counts was never reported, it appears that theta ie soma regional variation in mast-cell occurence which might, to a certain ezteat, parallel the variation in HA levels : mast-cells were, for instance, more frequently encountered in hypothalamus and thalamus, is which HA level is high, than in cerebellum, in which it ie low. Although these studies leave little doubt on the presence of mast-cells (or closely related cells) (IB, 2l) is mam®alian brain, they do mt, indeed, give a clue to the percentage of cerebral HA bald in these cells since this would require a precise knowledge of their HA content per cell, which is not available . However, recent data suggest that, at 1~~~t~ is the neonatal rat brain, msat-cells could well be the major store for HA . Although the level of moat cerebral amines is low at birth when synapses are few, it has long been a perplezing finding that, in contrast with the letters, the HA level ie almost eiz times higher than is the adult rat (4, 23, 24) (Fig . 1) . That this high level ie probably associated with mast-cells, is indicated, as zeeentl~ shown by Martree at al (22) by several lines of evidence : (a) a high level of HA, as is peripheral mast-calls, is accompanied by a low H .D. ac~ tivity (Fig . 1), thus suggesting that it ie turning over very e?~owly (HA in mast-calls l~ae been termed "dormant") ; (b) the half-life of H-HA formed is brain from S-histidiae injected at birth ie 4 days, a value close to that found is akin (a tissue rich in mast-cells) but contrasting with that in the adult brain (less than 1 h), (37) ; (c) after subcellular fractionation both

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Histamine as Transmitter in Brain

1

T

4

8

12

18 â0 Age (days)

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28

A~`

dulte

FIG I Developmental Pattern of HA and H .D . Activity in Rat Brain endogenous HA (44) and endogenously synthesized 3fl-HA are recovered is the de nuclear (P ) fraction, as the HA from typical mast-cells (isolated from peritoneal cavity) and added to brain homogenates (22, 26) ; (d) the two -cell degranulatora, compound 48/80 and Polymizine B, induce a significant lease of HA from incubated neonatal brain slices (22) .

cruthe mastre-

The picture appears different in the adult rat brain where there is axteneive evidence (s~oarized is Table I for the presence, in addition to the HA in mast-cells, of ßA in a neuronal system developing after birth . Fluorescence histochemical techniques which have been useful in the localization of central monoaminergic atones, although available for HA, have smfortunately been unable to visualize neuronal HA, probably due to lack of sensitivity (20, 21) . Different approaches have, however, provided indirect evidence for a HA-containing neuronal system . The subcellular distribution of HA in mammalian brain has been repeatedly investigated (26-31) . Although there are some discrepancies in the results (which are largely attributable to the differences is species, brain regions, schemes used for fractionation and methods for the HA assay) all reports conclude that a significant percentage of the amine ie present is the crude mitochondrial fraction . Thin fraction ie known to contain the eyaâptosomes ; i .e . the pinched off nerve-endings, and, when the letters are specifically isolated an a sucrose gradient, HA is, indeed, found is these particles . Furthermore, whoa synaptosomes are subjected to as hypoosmotic treatment, a substantial frac-

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TABLE I Tentative Synopsis of Differential Properties of the Histamine Stores in Neurones and Mast-Cells from Rat Brain .

PROPERTIES

SISTAMINE IN NEIIRONES

Subcellular localisation

aynaptoeomes (P 2B)

Appearance during brain maturation

late

Half-life Associated H.D . activity

(third week)

leas than

1 h

high

HISTAMINE IN MAST-(~LLS crude nuclear fraction (P 1) early (before birth) several days low

Effect of &+-induced depolarisation

released and syatfiesis accelerated

not released

Effect of reserpine

released and synthesis accelerated

not released

Effect of maat-cell degraaulators like Compound 48/80

not released

released

tion of the amine is recovered attached to synaptic vesicles (28, 30) . Although this pattern of distribution is suggestive of a neurotransmitter role for HA, its subcellular distribution differs from that of other putative transmitters by the praseace, in addition to the synaptoeomes, of a second eYase of storage particles . The latter were found to sediment either in the microsomal (P ) fraction (27, 28) or, in other studies, is the crude nuclear (P 1) fraction (~6, 31) (Table II) . This bimodal distribution was attributed by Rataoka and De Robertis (28) to the presence in rat torte: of histaminergic nerve endings of different sizes while for Ruhar et al (29), it was due to the radistributioa of endogenous HA during homogenisation . However, eves when this small redistributiôn artifact ie taken into account, ae is the data of Table II, it appears that the bimodal pattern persists and that a significant fraction of cortical HA is still sesociated with the crude nuclear fraction . Ia contrast with those of HA, the patterns for the aubcellular distribution and for the ontogenic developaent of the synthesizing enzyme were quite clear . A major fraction of the H.D . activity in adult rat brain waa recovered with the crude mitochondrial pellet (Table II) which density gradient centrifugation indicated to be associated with eynaptosomes (32) . Since this . .setivity could be released is soluble form by osmotic shock, it can be inferred that a major portion of the HA synthesizing enzyme is held in the cytoplasm of nerve terminals . This finding, together with that of HA is sgaaptic vesicles, obviously recalls the respective situations of other putative transmitters and their rate-limiting synthesising enzymes . The developmental pattern of H.D . (22) also reee~ bles closely that of enzymes like tyrosine hydrozylase, with an increase from birth to adulthood occurring with a time-course paralleling synaptogenesis (Fig 1) . Moreover, as in the teas of synthesizing enzymes for other putative transmitters, there woe, during brain maturation, a progressive shift in the subcellular localization of H. D. activity from the soluble to the syaaptosomal

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Table II ßubcellular Dia ribution of Bndogenoue HA, Bndogeaously Synthesized ~-HA and H .D . Activity in Rat Cortez .

Fractions

$adogenoue HA

z

~-HA

x

H.D . Activity

x

Nuclear (P 1)

33 + 1

14

12 + 2

Dtitochondrial (P2)

34 t 1

43

40 _+ 2

Microsomal (P 3)

17 + 1

,Supernatant (S 3)

17 + 1

43

40 + 3

8 + 1

The fractions were prepared according to Whittaker et al (1964), Endogenous HA (fluorometric assay) and H.D . activity (radiochromatographic assay) were measured in the same samples (mean + S .$ .ld. of 6 ezperimente) . The distribution of 3H-HA was determine~ in the brain of rate killed 1 h after intraveatricular administration of 'H-hietidine (37) . fraction (22) . In addition, Table II also summarizes the subcellular distribution of 3H-HA in the cortez of rats having received a tracer dose of H-histidine 1 h before sacrifice : the main difference with that of the endogenous amine was its lesser to calfzatioa in the nuclear (P ) fraction . Hence this fraction of HA in adult rat brain, sedimenting with the lcrude nuclear pellet, appears to be characterized by a low ratio of H.D . activity to endogenous HA level as well as by a slow turnover of ~-HA, all being characteristic of the amine stores in the neonatal brain ae well as in typical mast-cells . Furthermore, when typical mast-cells isolated from the rat peritoneal cavity were added to a brain homogenate before differential centrifugation, the HA was recovered almost entirely in the"crude nuclear" pellet (22, 26), suggesting that the amine in this fraction of brain is held in granules from cerebral meet-cells . Finally, the hypothesis of a dual localization of HA, is neurones and mast-cells, is also supported by data derived from in vitro ezperiments . Slices prepared from mouse brain (33) or rat hypothalamus 34 sad incubated in a physiological medium release both HA and endogeno~ely synthesized ~-HA (34, 35) when they are depolarised by the addition of R . As already shown for other putative neurotransmitters this release is calcium-depa~dent and, furthermore, it is accompanied bq a marked elevation in the rate of H-HA formation (35, 36) . Interestingly, peritoneal mast-cells do not release HA when they are incubated in a hi3gh R medium, suggesting that botä endogenous HA and endogenously synthesized H-HA released from depolarized brain slices probably arise from neurones and not from cerebral mast-cells . Oa the other hand, when a typical mast-cell degranulator, compound 48/80 is added o the medium in which hgpothalamic slices are incubated in the presence of ~-hietidine, ther3e ie a significant release of endogenous HA which occurs without release of H-HA egnthesized in the tissues (35, 36) . Again this apparent discrepaacq could be explained on the basis of a dual storage of cerebral HA ia, at least, tw compartments turning over at very different rates and whose differential properties are summarized is Table I. The rapidly turning over compartment has a half-life of a few minutes and

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is, therefore, selectively revealed by pules-labeling with the ~-precursor It is most probably neuronal because of its sub-cellular locali(15, 37, 38) . zation (37) and because of its sensitivity both to depolarization and to the action of neuropharaiacological agents like raeerpiae (39) or hypnotics (37, 40) . The slowly turning over compartment wind have a half- ifs of several days (22), would not ba, therefore, readily pules-labeled by the ~-preçursor and might be held is maet-cell granules which sediment in the crude nuclear fraction and are sensitive to liberators such as compound 48/80 or Polymizine B . This view ie consistent with the partial affects of synthesis inhibitors which induce a prompt fall of a fraction of cerebral HA (7, 8) but which leave intact a compartment representing about 50Z of the total, even if the drug dosage ie increased . Hence, in spite of the paucity of hiatochemical data available, the neurochemical approach indicates that HA-containing neurones must, indeed, be present in the CNS . Furthermore, lesion studies have recently thrown some light on the possible location of histaminergic pathways in brain . III :

Do Hietaminergic Neurones Contribute to the Medial Forebrain Bundle ?

The evaluation of the regional losses of putative transmitters and their synthesizing enzymes following selective brain lesions is e valuable tool in tracing neuronal pathways, especially when suitable hietochemical methods are not available . The first applications of this method to the localization of possible histaminergic pathways in brain have been made, using the level of HA as a neurochemical marker . There woe, however, no significant reduction of HA is brain regions following lesions in different meseacephalic or diencephalic structures (41) . Furthermore, large diencephalic lesions were unezpectedly found to cause a slow but marked elevation in hypothalamic HA in such a way that it could be tentatively attributed to a process of heterotypic reinaeroation (42, 43) . However, in the light of the esistence of a significant non-neuronal compartment for HA in brain, it appears that this interpretation might be questioned . In brain arses where inflammatory processes are likely to follow these large leeione, the appearance of non-neuronal HA-storing cells, like meet-cells, cannot be ezcluded . The processes wind perhaps resemble the increased number of meet-cells found in the sheathe of injured peripheral nerves . Bven more important, the considerations developed in the preceding section indicated that the neurochemical marker used in these studies was probably not specific for putative hietaminergic neurones and that the activity of H .D . would presumably represent a more reliable marker to be used in lesion studies . Starting from the observation of very similar patterns in the regional distribution of HA and the monoaminea aoradrenaline and serotonin, it was tempting to hypotheeige that histamiaergic fibers could be present in the Medial Fore brain Bundle (MFB), the ascending pathway which ie known to contain the bulk of the monoaminergic neurons . Therefore, dieacephalic leeione aiming at the interruption of this bundle ware performed uailatera4y at the level of the lateral hypothalamic area and their effects were checked both by histological ezamination and by measuring the ipeilateral decrease in cortical monoamines (noradrenaline and serotoain levels were decreased by 60-70X after 1 week (11, 12, 44) . Such leeione ware found to lover H .D . activity in the ipeilateral cortez, with a time-course compatible with a process of Wallerian degeneration . The enzyme activity in the contralateral aide was not significantly altered, indi-

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catiag that the telencephalic degeneration was restricted to the lesion side . The mazimal reduction in H.D . observed after 10 days was 50-60x but, at this time, the cortical HA content in the same animals was decreased by only 20-30x . Such a limited difference in HA content could ba detected only by comparing the endogenous amine level in the lesioned side to the corresponding intact side in the same individual animals and ezplains how it could be easily overlooked in other lesion studies in which HA was the only neurochemical marker used (41, 43) . The eziatence of a non-neuronal compartment of HA representing about 50x of the total amine in rat torte: and characterized, like mast-cells, by a low H.D . activity (cf . preceding section) could ezplaia why the lesions affected the synthesizing enzyme more than the level of amine . This view is supported by recent ezperimente is the cat in which the surgical isolation of a portion of the of the cortez (ia the aupraeylvian gyrue, in a way which does not impair the vascular supply but interrupts both the ascending and lateral afferent neurones) resulted in an almost complete dieappearaace of H .D, activity, whereas HA level was reduced only by 50x (45) . Similar distributions of HA- and monoamine-containing pathways in rat brain are indicated by the regional effects of the interruption of the MFB which affected all regions roetral to the lesion to a similar eztent, whereas enzyme activities in cerebellum, midbrain, pons and medulla were not significantly modified (Table III) . Although the lesions affecting HA is telencephalon also interrupted the moaoaminergic ascending pathways, their effect cannot be ascribed to an indirect consequence of the degeneration of the letters since neither 6-hydrozy-dopamine nor 5,6-dihydrozytryptamine did affect the H.D . activity in rat telencephalon (11, 12) . Hence, the eaieteace of a distinct hietaminergic pathway emanating from the midbrain or brain-stem, ascending through the lateral hypothalamic area and projecting in a widespread manner in the whole ipsilateral teleacephalon, appears vary likely . Such a disposition recalls that of the ascending monoaminergic pathways and suggests that, like the latter, the histaminergic fibers may not handle discrete information but set the overall suitability of telencephalon, perhaps in relay from the reticular formation, and could therefore participate in the control of states of wakefulness and sleep. A variety of data indirectly support this hypothesis . First, the fact that reserpine is able to release HA (34, 39) suggests that the mechanism of storage of the imidazoleamine might bear some similarity with that of the monoamines . In addition, several behavioural etuThe application of HA as wall as diea are in agreement with this hypothesis . that of its precursor amiaoacid in the lateral hypothalamus, has been shown to modify self-stimulation, a behaviour whose anatomical substrate is known to be the I4+B (46) . The participation of HA in cortical arousal mechanisms has also been evoked in view of the desynchronized E .E .G . patterns recorded after its The almost immediate decrease of cereintraveatricualr administration (47) . bral HA turnover in rate raceiviag barbiturates (37, 40), ae well ae the marked sedative properties of most aatihiatamiaes used in therapeutics (1), are also consistent with the possible involvement of ascending histaminergic neurones in arousal mechanisms . IV :

ßeçeptora to Histamine in Brais :

an Amine in Search of a Function

Although there ie now good neurochemical evidence for HA serving a transmitter function in the CNS, there are relatively few neurophysiological data for the involvement of histaminargic pathways in the control of specific neural functions or behaviours . Three complameatary strategies could theoretically be applied to reach this aim.

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Table III Modifi~tioas in L-hiatidine Decarbozylasa (H.D .) Activity in Regions of the Rat Brain after an IInilateral Lesion of the Medial Forebrain Btmdle .

Cortez Olfactory bulb Striatum Hippocampue Hypothalamus (anterior) Thalamus

-28 (± 5) ;

Meseacephaloa

+ 5 (+ 6)

Pons

+2 (+9)

Medulla oblongata

+25 (+11)

Cerebellum

+37 (+21)

The animals were killed 12 days after placement of the lesions at the level of the lateral hypothalamic area . Their localization was chec3ced by hietological azamiaatioa as well as by determination of the .cortical levels of noradreaaline ~ad aerotonia (decreased by 60-65x) . The table gives the mesa H.D . activity in the control side (dpm/~ug of tissue/h) sad the mean change (+ S.B .M .) of th lesioned aide compared to the control side in the same animal . 3 p 0.01 (I) The first approach consists is showing that the rate of HA release or, at least, of HA turnover in a given CHS structure is modified in relation with given physiological events . Indeed, neurochemical methods are already available to determine the rata of HA turnover, sad values of a few ainutea to a few hours have bean reported for the half-life of the amino in rat brain snider basal conditions (15, 37, 38) . Only in one instance, i .e . after a stress provoked by restraint or cold ezposure, was the rata of HA turnover fosmd to be modified, but the reported acceleration (10) could sot ba conf}rmd by others (48, 49) . (II) The second approach consists is evaluating the neurophyeiological or behavioural effects of drugs interfering specifically with the biosynthesis or inactivation of HA in brain. Unfortsmately, the chemical tools available until now lack selectivity, and no clear pattern has yet emerged from their utilization. For instance, HA biosynthesis can be enhanced by administration of the precursor aninoacid in high dosage (7, 8) . Such a treatment was reported to decrease the motor activity, especially in amphetamine-treated mice (50), sad to inhibit the self-stimulation behaviour in rats (46) . But the sigaificanca of these data is not clear, since hiatidine loads could modify ~on-histaminergic systems, i .e . by competition with the transport of other aminoacida . The use of potent inhibitors of HA synthesis also suffers from the same lack of

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selectivity since the hydraziao and oryamine derivatives, presently available kor this purpose, inhibit not only H.D . but other pyridoaal phosphate-dependent enzymes . (III) Finally, the third approach consists in evaluating the neurophysiological, neurochemical or behavioural effects of the ezogenous amine or of antâgoniste . This approach has been rather successful in providing evidence for the presence of HA receptors in mammalian brain. Furthermore, the two classes of HA-receptors, demonstrated (51) in peripheral tissues, i.e . the H -type (blocked by the classical antihistamines used in therapeutics) ând the l ~ type (not blocked by classical antihistamines and mediating, for instance tfie gastric action of HA) could also be identified in the C N S. The microelectrophoretical application of HA into the immediate vicinity of single neurons has been reported to alter resting potentials as well as firing rates. However, in a number of cases, the significance of the responses remains unclear since the strict conditions required, according to Bloom (52), for this kind of procedure, were barely met ; additionally, most of the studies have been performed before the discovery by Black et al . (53) of specific agonists and antagonists of H1-and H2-receptore, respectively . HA depressed the firing rate of manq interneurones and hyperpolarized motoneurones of the cat spinal cord (54) . A depressant, generally weak, action was also recorded after application of HA on neurones of the cuneate nucleus (55), brain stem reticular formation (56), motor precruciate cortex (57) or of the cerebellar Purkinje cells (58) . In contrast, the great majority of the cells in hypothalamus were suited by HA and, when HA was applied in high dosage on cortical neurones, ezcitation was also recorded (57) . The actions of HA on cerebral neurones appear to result from the activation of specific receptors ; they were not affected by the antagonists of other putative (:NS transmitters, whereas antagonists of H -receptors blocked partially or totally these effects. But the data with H antagonists should be considered with caution in view of their potent membraae l stabflizing action which results in a non-specific blockade of the actions of a variety of putative transmitters . The discovery by Kakiuchi and Ball (61) that HA was one of the most potent activators of cyclic AMP accumulation in brain slices has initiated a large set of studies which have already thrown some light on the nature of cerebral receptors to HA. It is clear that the effect of HA is mediated by receptors which are distinct from those mediating the raeponse of the cyclic AMP-generating system to noradrenaline . This is indicated by the large regional differences in respon siveness to the two biogenic amines, by the differences in sensitivity to the two amines during brain maturation (62) and, finally, by the lack of effect of ~(- and ~ -adrenergic receptor blockade on the HA-i~uced stimulation (63) . On the other hand, classical antihistamines, i.e . H -antagonists, have bean repeatedly found to inhibit the HA-induced stimulation, but, in moat cases, the blockade was either a partial one or vas complete only with high drug concentration~ . Recently, Baudry et al (65) demonstrated that the HA-induced accumulation of cyclic AMP in elicae from guinea-pig torte: was, in fact, mediated by the activation of the tw classes of receptors : it could be partially inhibited, in a dose-related fashion, by either a H -antagonist (mepyramina) or a H2 -antagonist (metiamide) and totally blocked iii the presence of both agents . Furthermore, a specific H2-agonist, 4-methglhietamine, induced a partial activation which, in this case, could be totally prevented by metiamide (Table IV) . the cellular localization (a) and functional role s) of the tw classes of

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Table IV fiffecta of Selective H 1 - and H2-Receptor Antagonists on the Activation of Cyclic AMP Accumulation in Slices from Guinea-Pig Cortez . ANTAGO~iIST3 OGONISTS None

Mepyramine

Metiamide

Mistamine

1+127x

+46x

+51X

4-Methyl-HA

~ +78x

+71x

N .S .

Mepyramine + Dletiamide N .S .

The values represent the percent increase in cyclic AMP accumulation âbove the aon-etimulated controls . N .S . refers to values non significantly different from those of the controls . cerebral HA receptors, evidenced by the stimulation of the cyclic AMP-ganeratiag system, is still a matter of conjecture . However, from their presence in the Cortez and hippocampua, i .e . regions where the lesion studies indicate that HA-containing neurones might be ending, it ie tempting to speculate that such receptors (or at least one kind of them) era localized post-synoptically sad that cyclic AMP ie playing the same role of "second messenger" at histaminergic synapses ae hypothesized at noradreaergic synapses of the cerebellum or hippocampus (52) . Consistent with this view are the recently reported (60) similar actions of cyclic AMP and HA which were microiontophoretically applied on cells of the cat medullary reticular formation : the great majority of calls depressed by cyclic AMP ware also depressed by HA. The finding of HA raceptore linked with the cyclic AMP-generating system on isolated nerva-eadinge (98) would also ba consistent with this hypothesis . On the other head, the responsiveness of cultured glioma cells (67) and,glia being the major morphological site of glycogen deposition, the finding of an H 1 -receptor-mediated stimulation of glycogenolysis in brain slices (68) would be in favour of a non-neuronal localisation of these receptors . The involvement of HA receptors is vegetative behaviour has been suggested by ezperimeats in which the eugenous amine was injected into specific areas or into the ventricular space . HA injected either into the roetral hypothalamus of the rat (69) or intraveatricularly in the mouse (70, 71) elicited as hypotherana which seems to be mediated both by H -and ~-receptors . Intrahypothalamic or intraventricular HA consistently elici~ed a wàter intake, as action blocked or even reversed by H -antagonists (72, 73) . Additionally, a marked aatidiureeie folloars the inject~oa of HA into the cat supraoptsc nucleus, which suggests that histaminergic neurones might be involved is the release of the antidiuretic hormone (74) . The emetic response elicited in the dog by stimulation of the chemoreceptor trigger zone is the area postrema ie mediated both by H 1 - and H2 -recaptor machaniems (75, 76) . Finally, the possible participation of ascending histaminargic fibers in

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Vol. 17, No . 4

the control of arousal mechanisms has previously been discussed (aectiaa III) . Since a participation of noradreaergic synapses in these mechanisms appears likely, the potentiation by HA of the aoradranaline-induced activation of the cyclic AMP-ganeratiag system in cortez (78) might be relevant . However, the nature of the receptors mediating the waking elicited by intraventricular HA is not clear ; the desynchronization of E.E .G . pattern has recently been reported to be reversed by low doses of peripherally-administered mepyramiae, an H -antagonist (79) . This antagonism was interpreted as waning that HA arou~al was mediated by peripheral receptore . but, since the effect of H -antagonists was not assessed, the interpretation probably deserves further consideration . y :

concluding Remarks

~pThen one ezamines the evidence for HA as a neurotransmitter is the mamma1$aa brain, it is rather convincing . A significant fraction of the cerebral amine is synthesized at a high rate and held in a specific neuronal tract, which can be released during depolarization and affects receptors mediating electrical, biochemical and behavioural effects . One of the aezt steps will be to identify more precisely the various histamiaergic synapses and to characterize the processes they might control . On the other head, it appears that another fraction of the cerebral amine might be held in non-neuronal cells, probably meet-cells or closely related cells . The function of mast-cells in the CNS, as in peripheral tissues, is far from clear but they might be involved in vascular control, immune responses or inflammatory processes. Interestingly, the mechanisms of HA release from mast-cells bear many similarities with the mechanisms of release of traasmitters from nerve endings (80) . Such a coincidence is probably not fortuitous, and suggests that during evolution a single convenient molecule might have been selected as a messenger acting in different kinds of cell-to-cell ao~uaications ; i .e . in neuronal, hormonal and immune processes. Acknowl e~emente It ie my pleasure to gratefully acknowledge the assistance of Pr M.J .Brody (Iowa-City) is the preparation of this manuscript and to thank M. de Boulongne for typing it so carefully . the work in the "Unité de Neurobiologie" ie granted by the following institutions t LN .S .E .R .M ., D . R.M.E . and F .R .M .F . . geferencas W.W. DOUGLAS, The Pharmacolo ical Basis of Thera eutice, 4~ edition, (Eds L.S . Goodman and A. Gilman , pp . 621-633, MacmllLan Company, London (1970) 2.

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