Epinephrine in mammalian brain

Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1988, Vol. 12, pp. 365-388 Printed in Great Britain.

0278-5846/88 $0.00 + .50 Pergamon Press plc

EPINEPHRINE IN M A M M A L I A N BRAIN

Ivan N. Mefford Laboratory of Clinical Science National Institute of Mental Health Bethesda, Maryland, USA (Final form, December, 1987) Contents 1. 2. 2.1 2.2 2.3 2.4 2.5 2.5.1 2.5.2 2.5.3 2.6 2.7 2.8 3.

Abstract Introduction Distribution of Epinephrine in Brain Phylogenetic Distribution of Epinephrine in Brain Regional Localization of Epinephrine in Mammalian Brain Ontogeny of Epinephrine and PNMT in Rat Brain Cellular and Subcellular l.x~calization of Epinephrine in Brain Tissue Presence of Epinephrine in Brain Extracellular Fluids Push-pull Perfusion Equilibrium Dialysis Cerebmspinal Fluid Epinephrine in Brain in Response to Stressors Pharmacological Manipulation of Epinephrine in Brain Epinephrine Synthesis and Storage Pools in Brain Significance and Conclusions Acknowledgements References

365 366 367 367 369 371 372 373 373 373 374 374 375 377 380 382 382

Abstract Mefford, I.N.: Epinephrine in mammalian brain. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1988, 12:365-388 1. Epinephrine is widely distributed in brains of various species throughout phylogeny but maintains its localization to hypothalamus and brainstem/medulla in all species studied. 2. A general decrease in brain epinephrine content is observed phylogenetically beyond fishes with wide variation within species. 3. The cellular localization of epinephrine forming enzyme is dissociated from epinephrine stores in hypothalamus where epinephrine appears to be primarily a hormone. 4. Three proposed functional pools of epinephrine are described. Synthesis of a hormonal pool and a second, perhaps nonfunctional, pool co-stored in noradrenergic terminals in the forebrain occurs extraneuronally and is probably inhibited acutely in the presence of high corticosteroids due to inhibition of uptake 2. Synthesis of epinephrine in the neuronal pool found primarily in the medulla may be enhanced due to increased PNMT activity in the presence of elevated corticosteroids. 5. Phylogenetic and pharmacological data suggest that epinephrine may play an important role in tonic regulation of the level of arousal, reward and sensitivity to environmental stimuli in mammals. Kevwords: arousal, brainstem, epinephrine, extraneuronal synthesis, hormone, hypothalamus, norepinephrine, phenylethanolamine-N-methyltransferase,'reward Abbreviations: Catechol-O-methyltransferase (COMT), Cerebrospinal fluid (CSF), N-2-chloroethyl-Nethyl-2-bromobenzylamine (DSP4), Dihydroxyphenylalanine (1-dopa), Monoamine oxidase (MAO), 6-hydroxydopamine (6-OHDA), Phenylethanolamine-N-methyltransferase (PNMT).

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1. Introduction Epinephrine was the first hormone to be isolated and crystallized (Oliver and Schaefer, 1895, Abel, 1902), being present in large quantities in adrenal extracts. In the intervening period a large body of scientific data has been collected concerning the hormonal role of adrenal epinephrine in the regulation of acute stress reactions, mobilization of glycogen stores, blood pressure, heart rate and respiratory interactions as well as several endocrine roles (Cannon, 1922, Holtz, 1948, Rothballer, 1959, Goldenberg, 1951). Early work identified epinephrine to be the primary catecholamine in frog or toad brain (Carlsson, 1959, Segura and Biscardi, 1967). Elucidation of the structure of "sympathin" in brain and peripheral nerves as that of norepinephrine in the 1940's (Vogt, 1954, Blaschko, 1942, Peart, 1949, von Euler, 1946) suggested that if epinephrine was present in mammalian brain it represented a minor fraction of the total catecholamine concentration. The work of Marthe Vogt (1954) and Lars Gunne in the early 1960's (Gunne, 1962) demonstrated that indeed epinephrine was present in mammalian brain although the concentration was quite low. In passing, it should be noted that the work of Vogt (1954) describing the presence and regional distribution of epinephrine in cat and dog brains represents an impressive analytical achievement. During the 1960's a number of groups were able to show that mammalian brain could synthesize labelled epinephrine from labelled norepinephrine (McGeer and McGeer, 1964, Barchas et al., 1969, Pahorecky et al., 1969). In the early 1970's with the advent of sensitive

radioenzymatic (Saavedra et al., 1974)

gas

chromatography/mass spectrometric (Koslow and Schlumpf, 1974) and liquid chromatographic (Keller et al., 1976) methods for analysis of minute quantities of catecholamines, several groups reported the heterogeneous distribution of epinephrine in rat brain (Saavedra et al., 1974, 1976, Koslow and Schlumpf, 1974, van der Gugten et ai., 1976). All of these data pointed to epinephrine synthesis in brain tissue distinct from uptake of blood borne hormone. The now classical description of the presence of neurons in mammalian brain capable of synthesizing epinephrine was made by Tomas Hokfelt and his collaborators in 1973 (Hokfelt et al., 1973, 1974). This observation was made possible through the development of a specific antibody for phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) by Menek Goldstein (Goldstein et al., 1974). Immunofluorescence histochemistry demonstrated the presence of neuronal networks in rat brain which contained immunoreactive PNMT, the enzyme necessary for the final biosynthetic step in the synthesis of epinephrine from norepinephrine. PNMT immunoreactive and presumptive epinephrine neurons were found throughout the midbrain, diencephalon, medulla and spinal cord of the rat (Hokfelt et al., 1973, 1974). Since these first descriptions of PNMT distribution in rat brain, the detailed distribution of epinephrine and PNMT have been described for several species (Goldstein et al., 1980, Mefford et al., 1977, 1978, 1981a, Beart et al., 1979, Kopp et al., 1979, Chamba and Renaud, 1983, Vogel et al., 1976, Lew et al., 1977, Kalia et al., 1984, 1985a,b). The effects of a fairly large body of "classical" pharmacological agents on epinephrine in brain have been performed (Fuller, 1982) and selective inhibitors of epinephrine synthesis have been developed (Fuller and Perry, 1977, Fuller et al., 1981, Pendleton et al., 1977a,b) led by the work of Ray Fuller and his associates. The present manuscript will review the literature, detailing the distribution of epinephrine in brain in various species and general phylogenetic trends in brain epinephrine content and PNMT activity. Pharmacological evidence both supporting and confounding the view of epinephrine as a classical

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neurotransmitter will be reviewed. Recent evidence of an extraneuronal pool of epinephrine will be discussed as well as the functional implications of this interpretation on the role of epinephrine in mammalian brain. 2. Distribution of Eoineohdne in Brain 2.1 Phvlo~enetic Distribution of EnineDhfine in Brain No systematic, comprehensive study of the phylogeny of epinephrine in the brains of various species has been performed. However, available literature, with some interpolation, does suggest several notable phylogenetic trends in epinephrine content or PNMT activity. Fuller and Hemfick-Luecke (1983) have surveyed epinephrine content and PNMT activity in hypothalamus and medulla of several species. This represents the most general survey of various species. Numerous investigators have published data on distribution in individual mammals, and while the high concentration of epinephrine in frog brain has been known for many years, the regional distribution within several amphibian subtypes was described by Cooney et al. (1985). Table 1. attempts to survey the available literature to provide as comprehensive a picture as possible of the distribution of epinephrine in brains of amphibians, reptiles, birds and mammals. The first notable trend in these data is the consistency of the regional distribution of epinephrine throughout these species. The hypothalamus and medulla contain the highest concentrations of epinephrine, regardless of the species examined. Cortical areas contain the lowest concentrations of epinephrine. There appears to be a general trend towards evolutionary elimination of epinephrine in brain as the concentration found generally decreases with increasing phylogenetic complexity. This is not observed as a smoothe or gradual decrease. Within each class which has been examined, there is considerable variability in epinephrine content. The few fish examined appear to have only small quantities of epinephrine in brain. Greatest epinephrine content is observed in frogs and toads in the class Amphibia and in tortoises in Reptilia. More than a five fold variation is seen between orders of Amphibia as is the case in the few reptiles examined. Epinepb_rine content generally exceeds norepinephrine in diencephalon in Amphibia, Epinephrine content also exceeds norepinephrine in tortoise midbrain and hypothalamus.

Turtles, on the other hand, have

norepinephrine in excess of epinephrine in hypothalamus and brainstem. Considerable variation is also observed in birds. The chicken brain contains the most epinephrine of those examined where epinephrine concentrations are 30 to 50% of norepinepbxine in hypothalamus. The goose brain contains roughly this same ratio of epinephrine to norepinephrine (M.M. Cooney and I.N. Mefford, unpublished observations). The pigeon, one of the more highly developed birds contains proportionally less epinephrine, about 5% of norepinephrine, in hypothalamus. Most widely studied are mammals, several species of which have been examined for both epinephrine content and PNMT activity. No obvious trend can be observed in terms of phylogenetic age. However, based on the few observations which have been made, some speculation can be made on general behavior or lifestyle and epinephrine content. Mammals which are carnivorous or display aggression in the face of environmental threat have fairly high levels of both epinephrine and PNMT activity. In general, mammals which are extremely vigilant and reactive, depending upon flight for survival instead of agression, have low epinephrine content and/or PNMT activity in brain. This is particularly notable for the guinea pig and rabbit,

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Table 1

Phylogenetic Distribution of Epinephrine in Brain, (ng/g tissue) SPECIE~

H__vpothalamus

Brainstem

CA~rebral (reference) Hemisoheres

Hagfish (Myxine glutinosa)

<20 (whole brain)

Cod (Gadus callarias)

30 (whole brain)

Goldfish (Carassius auratus) Hellbender (Chryptobranchus aUegeniensis) Mudpuppy (Necturus maculosus) Frog (Rana temboria) (Rana catesbeiana)

200.+ 90 1825.+ 24

179.+ 16

1110.+ 292

354.+ 31

190.+ 56

(Cooney et al., 1985)

4840.+ 720 1801.+ 101

740.+ 140 1214.+ 21

210.+ 90

(Jurio, 1973) (Fuller and Hemrick-Luecke, 1983)

Toad (Scaphiopus holbrookii) (Bufo terrestris) Turtle (Chrysemys picta)

2340.+ 188 2425.+ 311 323.

926.+ 68 670.+ 60 169.

876.+ 103 499.+ 41

(Cooney et al., 1985) (Cooney et al., 1985) (Fuller and Hemrick-Luecke, 1983)

Tortoise (Testudo graeca) (Testudo hermanni) Chicken (Gallus gallus)

4110.+ 560 2850.+ 280 100.+ 9.7

570.+ 100

170.+ 30 250.+ 30

650.+ 80 660.+ 70

400.+ 40

(Jurio, 1973) (JuNo, 1973) (Fuller and Hemrick-Luecke, 1983) (JuNo and Vogt, 1970) (Jurio and Vogt, 1970) (JuNo and Vogt, 1970) (Fuller and Hemrick-Luecke, 1983) (Dwyer et al., 1988) (Fuller and Hemrick-Luecke, 1983) (Fuller and Hemrick-Luecke, 1983) (Vogt, 1954) (Mefford et al., 1981a) (Fuller and Hemrick-Luecke, 1983) (Fuller and HemNck-Luecke, 1983) (Durkin et al., 1985) (Fuller and Hemrick-Luecke, 1983) (Fuller and Hemrick-Luecke, 1983) (Fuller and Hemrick-Luecke, 1983) (Caliguri, 1986) (Gunne, 1962) (Gunne, 1962) (Goldstein et al., 1980) (Mefford, 1986)

"Rhode Island Red" "Plymouth Rock" "Leghorn" Pigeon (Columba livia)

< 80. 72.+ 4

(von Euler and Fange, 1961) (von Euler and Fange, 1961) (Jurio, 1973) (Cooney et al., 1985)

52.+ 5.5

50.+ 6.4

210.+ 30 12.8 + 1.3

Tree Shrew (Tupai Tupai) Cat (Felis Catus)

30.to 438 79.+ 14

30.to 70 11.3 + 1.3

Dog (Canis familiaris)

76.+ 15

5.5 + 1.5

"Doberman" Ferret (Mustela putorius)

60.to 240 190.to 370 30.+ 2.0

12.to 200 8.2 + 0.8

Rat (Rattus norvegicus)

30.+ 2.1

6.9 + 0.4

10.to 30

2. to 5

Mouse (Mus musculus) Rabbit (Oryctolagus cuniculus)

7.2+2.0 ND.

1.3+0.1 ND.

ND ND.

Guinea Pig (Cavia porcellus)

ND.

ND.

ND.

Horse (Equus caballus)

ND.

ND.

ND.

Ox Pig Monkey (Cerapithecus Sabaeus) Human (Homo sapiens)

0. tol0 0.to 6 ND. ND 7.5 % of norepinephrine 52.to 450 6.to 160 < 5.0 23.to 136 0.to 45 0.4 to 1.1

very and to a lesser extent, several herbivores. The adrenal gland in these animals contains almost entirely epinephrine as was noted some thirty years ago (Holtz, 1950, Goldenberg, 1951). This suggests a possible

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369

inverse relationship between epinephrine in brain and adrenal with high epinephrine in adrenal and low epinephrine in brain associated with high arousal and reactivity to environmental threat. Interestingly, there is not a one to one correspondence between epinephrine content and PNMT activity (Fuller and Hemricke-Luecke, 1983) although they are reasonably correlated. Goldstein (Goldstein et al., 1980) has observed that the distribution of both PNMT and epinephrine is quite wide in the primate brain, suggesting perhaps a more prominant role in this species than in some mammals. Although we have published fairly widespread distribution of epinephrine in human brain, and relatively high concentrations (Mefford et al., 1978, 1986) observations made on autopsy specimens obtained within 45 minutes of death suggest that endogenous epinephrine content in human hypothalamus might be somewhat lower than previously suspected (I. Mefford, K.A. Roth and J.D. Barchas, unpublished observations). It has not been established whether epinephrine plays ~lay role in the central nervous system in "arousal", however, pharmacological evidence of possible involvement of central epinephrine in these phenomena will be addressed in a later section. 2.2 Reeional Localization of EoineDhrine in Mammalian Brain The earliest descriptions of epinephrine distribution in rat brain showed highest concentrations to be in the hypothalamus and medulla and in general to be distributed throughout the midline areas of the brain. As was suggested by the distribution of PNMT activity and immunofluorescence, relatively high concentrations of epinephrine were found in the ventral lateral reticular formation in the cell body areas denoted C1 and in the dorsal areas denoted C2 and the more recently described C3 (Howe et al., 1980) More detailed distribution of epinephrine can be studied in the brains of larger mammals. Both the dog and human brain have been studied (Mefford et al., 1977, 1978, 1981a, Mefford, 1986). These data are summarized in Table 2. All values presented are ng/g tissue wet weight and were obtained using high performance liquid chromatography with amperometric detection (Mefford, 1985). Several points are notable concerning the distribution of epinephrine in these brains. The correlation between the distribution of epinephrine and norepinephrine is > +0.8 in both species. Highest concentrations are found in the various hypothalamic nuclei. Within the hypothaiamus, highest concentrations are found in the most medial areas, peri- and paraventricular and dorsomedial hypothalamus.

Anterior preoptic

hypothalamus is also rather rich in epinephrine content. Proceeding laterally, the concentration of epinephrine decreases markedly. Very low concentrations of epinephrine are found outside the lateral confines of the hypothalamus. Proceeding rostrally, the concentration of epinephrine again decreases markedly outside the anterior/preoptic hypothalamus where highest concentrations of epinephrine are found in the septal nuclei and in the nucleus accumbens. Moving dorsally and laterally, epinephrine content again decreases markedly with very little epinephrine found in the caudate, putamen, globus pallidus, hippocampus or cortex. Caudally from the hypothalamus, epinephrine content remains relatively high, especially in midline areas near the ventricles. Aqueductal grey remains consistently high in epinephrine content throughout the brainstem. The area around the floor of the fourth ventricle near the C2 and C3 cell body areas contains relatively high epinephrine concentrations as does the pontis oralis and pontis gigantocellularis of the lateral reticular formation. Only the most rostral portions of the spinal cord were analyzed, however, histofluorescence data from the rat suggests that epinephrine will be found throughout the spinal cord in reasonably high concentrations (Jonsson et al. 1976, Ross et al., 1981). The cerebellum was comparable to or less than the cerebral cortex in epinephrine content. Perhaps the most apparent observation concerning the distribution of epinephrine in brain is its

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I.N. Mefford

Table 2 Distribution of Epinephrine in Dog and Human Brains

Doe CORTEX G. Rectus G. Presylvius G. Coronalis G. Cruciatus G. Frontalis G. Cingulate THALAMUS N. Anterovent. N. Med. Dors. N. Vent. Lat. N. Pulvinaris Subst. Grisea Centralis Lateral Geniculate Medial Geniculate Area Septalis Hippocampus Amygdala Dorsal Ventral Cerebellum Substanfia Nigra BASAL GANGLIA N. Caudatus Ant. N. Caudatus Med. N. Caudatus Caud. Putamen Globus PaUidus N. Accumbens Septi HYPOTHALAMUS Area Preoptica N. Dorsomedialis N. Ventromedialis N. Posterior N. Lateralis BRAINSTEM/MEDULLA N. Hypoglossi N. Olivaris inf. N. Occulomotorii N. Ruber N. Vestibularis Inferior Colliculus Superior Colliculus Subst. Grisea Centralis N. Tractus Solitaris N. Retic. Pontis Oralis N. Retic. Pontis Caudalis N. Retic. Pontis Gigantocell. N. Retic. Ventralis N. Centralis Superior N. Dorsalis Raphe N. Raphe N. Abducentis N. Praepositus

3.6 2.0 4.5 1.6 3.6 3.6

Human

+ + + + + +

0.6 1.4 1.7 0.4 1.4 1.7

---0.4 to 1.1 ---

12.8 + 1.3 15.4 + 2.1 14.9 + 11.7 5.4 + 1.9 31.7 + 15.8 6.0 + 2.1 2.8 + 0.4 43.5 + 30.8 2.1 + 1.0 6.8 + 2.9 19.1 + 11.5 5.2 + 2.6 3.1 + 0.2 10.6 + 0.4

1.5 14.2 + 6.0 4.3 + 1.6 6.2 + 2.3 10.6 + 3.2 --49.7 + 17.3 0.9 + 0.4 ---< 0.2 4.3 + 2.2

ND ND ND ND 26.0 + 12.0 56.0 + 30.0

-ND to 0.6 -ND to 0.1 0.6 9.2 + 4.1

367. 204. 447. 178. 193.

115 26 211 153 54

68. + 20 106. + 39 136. + 55 33. + 9 23. + 9

16.1 & 7.1 55.4 + 6.5 126. + 84 202. + 100 26.6 + 9.7 8.5 + 2.5 11.4 + 6.1 117. + 39 48.9 + 7.5 27.8 + 1.2 73.6 + 8.2 21.7 + 7.6 26.8 + 3.3 92.8 + 20.5 42.6 + 8.7 45.5 + 15.6 12.8 + 0.8 22.0 + 8.8

35.to 45 ND. to 2 9.8 + 3.2 4.7 + 2.3 5.to 10 ND.to 5 5. 20.to 40 35.to 45 20 35.to 45 20 10.to 20 10.to 20 20.to 25 10.to 20 5.to 10 10.to 20

+ + + + +

(summarized from Mefford et al., 1977, 1978, 1981a, Mefford, 1986. All values ng/g tissue. ND not detectable, -- = not measured.)

Epinephrine in brain

localization to the phylogenetically more primitive areas of the brain. This suggests a higher probability of a functional role in primitive behaviors or regulation of primary functions rather than direct regulation of higher cortical or cognitive functions. 2.3 Onto~env of Enineohrine and PNMT in l~at Brain In addition to the suggestions of function which can be derived from the regional localization and phylogenetic distribution of epinephrine, the ontogenetic development of PNMT and presence of epinephrine in developing brain might be indicative of function. Only a few studies addressing this topic have been accomplished. The presence and postnatal development of epinephrine in the rat brain was studied by Milby et al. (1979). The concentration of epinephrine, norepinephrine and dopamine was measured in midline core areas of rat brain, containing the hypothalamus, thalamus, colliculi, pons and medulla, but lacking the cerebral hemispheres, swiata and cerebellum. A gradual rise in epinephrine content was observed from day birth through day 15 or 16 where a maximum was observed. After a slight decrease between day 17 and 22, no change was observed through adulthood. PNMT activity was not measured. Foster and coworkers (Foster et al., 1985) studied the prenatal development of PNMT-like immunofluorescence. They observed that at day 13 of gestation, PNMT-like immunofluorescence appeared throughout the medulla and hypothalamus.

Other biosynthetic enzymes were not observed

immunohistochemically in the medullary cells until after day 16, and were not observed at all in the hypothalamus. PNMT-like immunofluorescence was not notably different at birth than at day 13 of gestation. The failure to observe the presence of other biosynthetic enzymes at this stage of development suggested that PNMT might be capable of synthesizing some N-methyl compound other than epinephrine. These authors, in a follow-up study, examined the presence of epinephrine, norepinephrine and dopamine and PNMT and Tyrosine Hydroxylase activities measured in vitro in the dorsal and ventral medulla in prenatal and adult rat brain (Foster et al., 1987). PNMT activity was slightly increased between day 14 of gestation and adulthood in the ventral medulla (primarily the C1 cell group) while PNMT activity at adulthood was only about 30% of the day 14 PNMT activity in the dorsal aspects (primarily the C2 and C3 cell groups). Epinephrine content was not markedly increased at adulthood over day 14 of gestation in either dorsal or ventral aspects. While the epinephrine represented 30 to 50% of the total catecholamines in this / area at day 14 of gestation, the marked increase in norepinephrine content through development had lowered this contribution to 2-3% by adulthood. Bohn et al. (1986) performed a similar study, however, using total catalytic activity rather than normalizing to protein or tissue weight. They showed a gradual increase in PNMT activity in medulla through postnatal day 20 and little change thereafter. These data show the divergence in the development of epinephrine synthesizing capacity of epinephrine and norepinephrine in the developing brain. The relative presence of epinephrine is much greater in early stages of development in the rat. This suggests a parallel between ontogeny and phylogeny, although there is no evidence that the functional significance of epinephrine is greater at any particular stage of development. The relative contribution of epinephrine to total catecholamine content appears to decrease with increasing activation during ontogeny. Comparing the data of Milby et al. (1979) and Foster et al. (1987), it would appear that the presence of epinephrine in parts of the developing rat brain other than the medullary areas, C1-C3, develops more slowly, even though PNMT-like immunofluorescence is present, and is dependent upon the development of norepinephrine neurons or norepinephrine synthesizing capacity. It is also possible

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I.N. Mefford

that the development of epinephrine content in these other parts of the brain is dependent upon the expression of an N-methyltransferase other than PNMT. The data of Bohn et al. (1986) appear more consistent with the observations of Milby et a1.(1979), showing a gradual increase posmatally in epinephrine synthesizing capacity. 2.4 Cellular and Subcellular Localization of EDineohrine in Brain Tissue Evidence for the cellular subtype and subceUular localization of epinephrine is derived from two types of experiments. Selective lesioning of catecholamine neurons using DSP-4 or 6-hydroxydopamine (6-OHDA) or surgical transection of presumed ascending projections have provided evidence of the neuronal localization of epinephrine. Subcellular fractionation and differential centrifugation have been used to show the intraneuronal and granular localization of epinephrine. No selectivity for uptake of epinephrine versus norepinephrine has been observed in rat hypothalamus (Routledge and Marsden, 1987c). Jonsson et al. (1976) and Reid et al. (1976) demonstrated in the mid 1970's that PNMT containing cells were resistant to the neurotoxic effects of 6-OHDA. Numerous investigators have since confirmed these results (Mefford, 1987a, Cooney, 1986, Renaud et al, 1986), simultaneously demonstrating that epinephrine concentration was markedly depleted by these lesions. These results demonstrated the dissociation between PNMT activity and the site of storage of epinephrine in brain. Jonsson suggested that neurons which synthesized epinephrine contained no specific high affinity uptake site (uptake 1) for 6-OHDA and that further, locally released norepinephrine might serve as substrate for epinephrine synthesis, rather than norepinephrine synthesized in the PNMT containing neuron (Jonsson et al., 1980). DSP-4 represents a similar but more selective type of neurotoxin for causing degeneration of noradrenergic terminals. Again, Jonsson and coworkers (Jonsson et al., 1981, Durkin et al., 1985) and others (Renaud et al., 1986) used this neurotoxin to study storage of norepinephrine and epinephrine in rat and mouse brain. These data indicate that this neurotoxin, which selectively causes degeneration of locus coeruleus noradrenergic projections, does not affect the sites of storage of epinephrine. This implies that the noradrenergic projections with which colocalization of epinephrine is associated arise not from the locus coeruleus but the brainstem A1 and A2 cell groups. Surgical procedures have been used to isolate the nerve terminal regions (hypothalamus and spinal cord) from the cell body and PNMT containing areas in the medulla (Brownstein et al., 1976, Palkovits et al., 1980). Hemitransections in which unilateral ascending projections are severed lead to marked depletion of epinephrine and norepinephrine the terminal fields of these projections in hypothalamus (Mefford et al., 1981b). PNMT activity is unaffected (Saavedra et al, 1983). Transection of the descending projections from the medulla leads to depletion of epinephrine and norepinephrine as well as a decrease in PNMT activity (Reid et al., 1976). Further, adrenal demeduUation has no effect on epinephrine concentrations in any brain region studied (Mefford et al., 1981b). These data confL'm the observations made after chemical lesions of catecholamine terminals in the hypothalamus, that the epinephrine present is dissociated from the PNMT activity. This dissociation suggests that the bulk of epinephrine found in the hypothalamus is present in noradrenergic terminals and storage vesicles while PNMT is present in another type of cell or neuron (Mefford, 1987a, Ross et al., 1984 ). Thus, the bulk of N-methyltransferase activity in the hypothalamus appears not to be associated with the ascending projections of the C1-C3 cell body groups.

Epinephrine in brain

Subcellular fractionation of brain tissue confirms the presence and localization of the bulk of epinephrine in both hypothalamus and brainstem in storage granules (Jonsson et al., 1981). The distribution in subcellular compartments is almost identical for epinephrine, norepinephrine, and dopamine in both regions. PNMT activity is found primarily in the soluble fraction while a small fraction may be membrane bound (CiaraneHo et al., 1969). Finally, it must be noted that PNMT has been found in pericytes and endothelial cells cultured from brain capillaries (Spatz et al., 1979) as well as in ganglia (Moore and Phillipson, 1975, Phillipson and Moore, 1975). These data suggest that not all the PNMT or epinephrine measured in brain is necessarily of CNS origin, but may arise from sympathetic innervation or from synthesis in other types of cells. No contribution of adrenal epinephrine to hypothalamic epinephrine content has been observed in rat brain (Mefford et al., 1981b). 2.5 Presence of Eninenhrine in Brain Extracellular Fluids Given the low concentration of epinephrine in brain tissue, one would anticipate very low concentrations of epinephrine in extracellular fluid and cerebrospinal fluid. Under most circumstances this is the case for cerebrospinal fluid. However, several investigators have noted surprising quantities of epinephrine in extracellular fluid in hypothalamus using two different collection techniques. 2.5.1 Push-null Perfusion, These techniques have been used for many years to study the release of endogenous amines in unanesthetized and anesthetized animals. Data obtained from perfused hypothalamus using this technique show quantifies of epinephrine in exlracellular space which are not consistent with tissue content. Phillipu and coworkers (Phillipu et al., 1979, 1980, 1981) have demonstrated the efflux of roughly equivalent concentrations of epinephrine and norepinephrine in perfusate from hypothalamus. This observation has been made in several species with widely varying tissue epinephrine content, the rabbit, rat and cat respectively (see table 2). Hypothalarnic epinephrine in these species varies from less than 1% to as much as 20% of norepinephrine. One might question these data based on the method of quantitation, however, similar results are obtained using equilibrium dialysis and a different analytical technique for determination of epinephrine in the perfusate. Some ten years ago we observed similarly high proportions of epinephrine in push-pull perfusates from rabbit hypothalamus following ethanol administration (I.N. Mefford, R. Huff, C.K. Erickson and R.N. Adams, unpublished observations). We considered these measurements obvious artifacts inconsistent with the low tissue epinephrine content, perhaps a foolish conclusion, and abandoned the experiments. 2.5.2 ~quilibrium Dialvsis. Routledge and Marsden (1987a,b) have demonstrated the presence of epinephrine at approximately 70 nanomoles/liter in extracellnlar fluid via dialysis from the posterior hypothalamus even following adrenal demedullation. This concentration is only modestly less than that observed for norepinephrine in these experiments, even though epinephrine tissue content is only 1-4% of the norepinephrinc concentration in this area of rat brain. These researchers have examined effects of a number of pharmacological treatments on cxtraueuronal epinephrine concentration. These data are quite intriguing and suggest that based on the tissue content of epinephrine, the turnover must be quite high in

373

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epinephrine containing neurons in order to maintain these high extraeellular concentrations. Using a variety of pharmacological tools, however, it can be shown by classical enzyme inhibition measures, that the turnover of hypothalamic epinephrine stores is quite similar to norepinephrine, with tl/2 on the order of 1.5 to 3.0 hours (Sauter et ai., 1977). Routledge and Marsden have demonstrated that following PNMT inhibition with LY87130, extracellular epinephrine is depleted much more rapidly than is tissue content (1987b). These data suggest that two different pools of epinephrine are measured by these methods. Tissue content measurements reflect a pool with relatively slow turnover comparable to that of the granular noradrenergic neuronal pool. The extracellular measurement represents a pool with a tl/2 of considerably less. As was recently hypothesized, this extracellular pool may represent the functional pool of epinephrine, which may function as a hormone (Mefford, 1987). Using similar equilibrium dialysis techniques, we have observed extracellular fluid concentrations of unconjugated epinephrine to be less than 3 nanomolar in several hypothalamic areas (J. Hsiao, R. Barraco, W.Z. Potter and I.N. Mefford, unpublished observations). Rat brain tissue measurements represent primarily epinephrine co-stored in noradrenergic neurons. The bulk of this may represent a nonfunctional pool, an artifact of the specificity of noradrenergic terminals for catecholamine uptake (Routledge and Marsden, 1987c), as is the case for the bulk of epinephrine found in peripheral sympathetic temainals (Sudo, 1985, Saavedra et al., 1982). 2.5.3 Cerebrost~inal Fluid. CSF represents a more integrated measure of extracellular amines and metabolites. Few reports have appeared of basal levels of epinephrine in CSF. Christensen has reported mean values of more than 20 pg/ml in human CSF (Christensen et al., 1980). We reported mean values of no more than 10 pg/ml (Berger et al., 1984). Using much more sensitive and selective analytical techniques (Durkin et al., 1985, Mefford et al., 1987a) we have now measured epinephrine in a variety of clinical populations. Epinephrine concentrations in untreated controls are less than 5 pg/ml and frequently not detectable, less than 1 pg/ml (I.N. Mefford, D. Pickar and W.Z. Potter, unpublished observations). Similar results have been obtained in CSF obtained from chair adapted Rhesus monkeys (Mefford et al., 1987b), with mean basal values of approximately 5 pg/ml. Epinephrine found in CSF is found in both free and conjugated forms (Ratge et al., 1985). This suggests that a fraction of the extraneuronal pool may be in the conjugated form and that conjugation via phenol sulfotransferase might be a significant clearance mechanism for this pool. 2.6 Eoinenhrine in Brain in Response to Stressors One of the earliest observations with respect to hypothalamic epinephrine was its lability to stressors. Several authors have observed that acute stress, whether mild or strong, can induce marked depletion of hypothalamic epinephrine (Swible-Keane, 1986, Mefford, 1987a, Roth et al., 1982, Kvetnansky et al., 1978, Saavedra et al., 1980, Sauter, et al., 1983, Beart, 1979, Mefford and Swible-Keane, submitted). The depletion of epinephrine is not matched by the depletion of norepinephrine, which is either not depleted or only modestly depleted by these same stressors (Roth et al., 1982). In addition to marked lability of hypothalamic epinephrine content in response to stress, the replenishment of epinephrine is much slower than that of norepinephrine (Mefford, 1987a, Swible-Keane, 1986, Roth et al., 1982). Both of these observations are consistent with the dissociation of the site of synthesis and the site of storage of epinephrine in hypothalamus. Brainstem/medullary epinephrine is much less labile in response to stress. Only infrequently are stressors severe enough to cause marked depletion of epinephrine. When this does occur, recovery is rapid and similar to norepinephrine. The ability of a stressor to deplete hypothalamic epinephrine

Epinephrine in brain

does not appear to be substance or stress specific. Numerous chemical agents, not necessarily involved directly in synthesis of epinephrine have been shown to cause depletion of hypothalamic epinephrine (Fuller, 1982), including ethanol (Mefford, 1987b, Mefford et al., submitted a), nicotine (Roth et al., 1982), morphine, picrotoxin (Vogt, 1954), insulin (Sauter et al., 1983, Swible-Keane, 1986) and dopamine agonists (Fuller, 1982, Fuller and Hemrick-Luecke, 1985). The response of the synthetic enzyme, PNMT, to stressors is quite different. Brainstem PNMT is elevated by both physical and physiological stressors (Turner et al., 1978, Saavedra et al., 1976, 1980, 1983) and the administration of glucocorticoids (Turner et al., 1979, Moore and Phillipson, 1975). Hypothalamic epinephrine is relatively unaffected by these manipulations. Moore and eoworkers have demonstrated that ganglionic PNMT is markedly elevated following dexamethazone administration (Moore and Phillipson, 1975, Phillipson and Moore, 1975). This may be mediated by the same mechanism as in the adrenal medulla, where glucocorticoids induce new protein synthesis and stabilize PNMT, effectively increasing the enzyme lifetime (CiaraneUo, 1978). The measurement of epinephrine content in response to stressors indicates that the tissue pool, particularly in the hypothalamus, is easily depleted by stress. This might reflect the site of storage of this pool, primarily in noradrenergic neurons, dissociated from the site of synthesis. These measurements do not address the response of extracellular epinephrine in response to stress. Routledge and Marsden have demonstrated a marked increase in extracellular epinephrine in response to electrical stimulation of the C1 region (Routledge and Marsden, 1987a). Phillipu and coworkers have demonstrated that extracellular epinephrine increases markedly in response to a number of physiological stressors including severe hypotension (Phillipu et al., 1979, 1980, 1981). Epinephrine efflux has been observed in portal blood in adrenalectomized rats in response to several stressors (Gibbs, 1985, Johnston et al., 1983). Finally, Torda (1977) has observed the marked accumulation of epinephrine in hypothalamus following electrical stimulation of the medial forebrain bundle, when alternate metabolic pathways for norepinephrine were blocked. All of these data point to a marked increase in extraneuronal epinephrine in response to stress. It should be noted that the release of epinephrine observed by Routledge and Marsden, as well as that observed in portal blood was not accompanied by increased norepinephrine efflux. 2.7 PharmacoloL,ical Maninulation of Eninenhrine in Brain Fuller has published a rather comprehensive review of the pharmacological manipulation of brain epinephrine (Fuller, 1982). This section will address only those compounds shown to markedly affect tissue or extracellular epinephrine. The most selective method of altering epinephrine content is of course to selectively inhibit its synthesis. A variety of inhibitors of PNMT have been developed which can be used in animal studies. All of these compounds share structural features with similar compounds which are potent alpha-2 receptor antagonists or monoamine oxidase inhibitors (Fuller et al., 1981). As a result, behavioral or neuroendocrine effects of these compounds must always be posited in these terms. The complete elimination of tissue epinephrine following PNMT inhibition is quite difficult, but can be accomplished when stress or reuptake blockade is superimposed.

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PNMT inhibitors have a variety of behavioral effects. If these effects are the products of central PNMT inhibition and not some side effect of these various drugs, then a significant role for epinephrine is implied in several behaviors. Acute, observable effects of several PNMT inhibitors, including SKF64139, LY134046 and LY78335, are tremor, sniffing, burrowing and general activation in the home cage. These effects are qualitatively similar to ethanol or opiate "withdrawal" syndromes (Mefford et al., submitted b). This is consistent with the observation of withdrawal after the cessation of intraventricular epinephrine infusion

(Jeffries and Orzechowski, 1985). PNMT inhibition enhances the motor effects of amphvtamine (Katz et al., 1978a), causing additional activation. Open field activity, on the other hand, is decreased by PNMT inhibition (Katz et. al, 1978b). Katz and coworkers have observed marked inhibition of intracranial self-stimulation following administration of PNMT inhibitors, an effect also observed after the administration of yohimbine (Katz and Carroll, 1977, Katz et. al., 1978c, 1979). We have recently observed almost complete antagonism of ethanol-induced intoxication following administration of PNMT inhibitors (Mefford et al., submitted b). Chronic administration of PNMT inhibitors leads to profound changes in the alpha-2 adrenergic receptor number in rat brain (Stolk et. al., 1984). When considered with the evidence that alpha-2 receptor number is inversely correlated with PNMT activity in inbred rat strains (Perry et al., 1983, Vantini et al., 1984), these data suggest that epinephrine might serve as an important agonist at this receptor subtype in rat brain. Routledge and Marsden have demonstrated that PNMT inhibition eliminates extracellular epinephrine even though tissue content is not totally depleted (Rontledgc and Marsden, 1987a). The effects of other drugs am less selective for epinephrine than the inhibitors of PNMT. In fact, present evidence suggests that the effects of most drugs which affect nompinephrine release or rnctabolism have a similar effect on epinephrine release and metabolism. Some selectivity towards epinephrine has been observed with inhibitors of monoamine oxidase type A (Mefford et al., 1985, Gjerris et al, 1984). In vitro evidence suggests that epinephrine is an excellent substrate for MAO t y ~ A (7_cllerand Arora, 1979). Fuller has demonstrated that MAO type A is the endogenous enzyme for oxidative deamination of epinephrine (Fuller and Hemrick-Luecke, 1980, 1981). Chronic administration of LY51641, a selective inhibitor of MAO type A, causes a disproportionate increase in brain epinephrine compared to nompinephrine (Mefford et al., 1985). Both epinephrine and norepinephrine am increased to the same extent acutely in exwacellular fluid following administration of tranylcypromine (Roufledge and Marsden, 1987b). All of these data demonstrate that metabolism of both epinephrine and norepinephrine by MAO type A represents a major route of elimination. The only other selective manipulation of epinephrine in tissue is accomplished with administration of l-Dopa. Fuller and coworkers, in a series of papers, have examined the antagonism of the MAO-induced increase in hypothalamic epinephrine by l-Dopa (Fuller et al., 1982a) and depletion of epinephrine by l-Dopa administration. Initially it was hypothesized that this effect might be duo to excessive formation of S-adenosylhomocysteine following O-methylation of 1-Dopa, which could serve as an inhibitor of PNMT (Fuller et al., 19821)). Further experimentation has demonstrated that this is not the likely mechanism as co-administration of an inhibitor of COMT, tropolone, leads to a much more marked reduction of epinephrine (Fuller et al., 1984). An alternate explanation of these observations is that epinephrine in hypothalamus is primarily an extraneuronal metabolite of norepinephrine. In such a case, administration of 1-Dopa would shift the equilibrium for filling storage sites toward intraneuronal synthesis and away from uptake of extraneuronal material (Mefford, 1987a). This interpretation is also consistent with the further

Epinephrine in brain

depletion following tropolone administration. 2.8 Eninenhrine Synthesis and StoraCe Pools in Brain In a recent review a hormonal role was proposed for the functional pool of epinephrine in hypothalamus and probably a fraction of the epinephrine in the brainstem/medulla (Mefford, 1987a). Under that proposal, epinephrine in brain exists in three different pools. The bulk of that which is measured in traditional tissue measurements is found co-stored with norepinephrine in noradrenergic terminals. This is likely for most of hypothalamic epinephrine and probably a reasonable fraction of brainstem/medulla epinephrine. Also in the brainstem are numerous neurons containing all the necessary biosynthetic enzymes for the de novo synthesis of epinephrine intraneuronally (Foster et al., 1985, Kalia et al., 1984, 1985a, 1985b, Ruggiero et al., 1985, Ross et al., 1981). These neurons represent the true "epinephrine" neurons, ff such a beast exists. The third pool which is likely found throughout the medial aspects of the brain and spinal cord is found in extracellular fluid and represents epinephrine formed in an ex~aneuronal site containing no storage sites. The site of this synthesis is at present unknown, but may be in "non-catecholamine" neurons or perhaps in a population of glial cells (Mefford et al., 1987b), pericytes or endothelial cells (Spatz et al., 1979). The failure of the PNMT-containing cells to degenerate in response to intracerebral administration of 6-OHDA is reminiscent of similar observations with adrenal chromaffin cells and the small intensely fluorescent (SW) cells of the neonatal Superior cervical ganglia, a population of cells in which PNMT can also be induced by corticosteroids (Gianutsos and Moore, 1977). Liposits et al. (1986) recently described granule-containing cells in the paraventricular hypothaiarnus, also containing PNMT, presumed to be neurons. The functional significances of these three pools are likely quite different. Data reviewed earlierin this text of high extracellularepinephrine concentrations, roughly 10 -7 M (Routledge and Marsden, 1987a,b), and that reviewed earlierfor marked synthesis during stimulation (Torda, 1977, Gibbs, 1985, Johnston et al., 1983, Phillipu et al., 1979, 1980, 1981), suggest that epinephrine can be formed in relatively high quantities in extraneuronal sites. This extracellularepinephrine which probably represents a hormonal pool may be the most important pool, at least in the forebrain. Such a pool could provide tonic inhibitory regulation of neuronal activitythrough nonsynaptic alpha-2 receptors CU'Prichard et al.,1980, Svensson et al., 1975, Cedarbanm and Aghajanian, 1976) and would represent a mechanism for transduction of neuronally released norepinephrine via a second cell. As is the case with exogenously administered alpha-2 agonists which are anxiolytic and sedative (Washton and Resnick, 1980, Gold et al., 1979, Drew et al., 1979), the epinephrine in thispool would provide a mechanism for regulating the general level of arousal. Figure 1. shows the hypothetical arrangement of noradrenergic terminal and storage granules and the dissociated PNMT-containing cells in forebrain which could regulate epinephrine formation in a hormonal pool. The content of this pool should be responsive to release of norepinephrine in surrounding neurons. As a result, the concentration of epinephrine in this pool would be dependent on substrate availability (norepinephrine released from surrounding neurons), PNMT activity in the exWaneuronai sites and uptake of norepinephrine into exwaneuronal sites. The uptake site on these "non-catecholamine neuron" cells is likely to be similar to the steroid sensitive uptake-2 site described by Iverson (1965) and Salt (1972). As a result, during times of high conicosteroid or glucocorticoid output, epinephrine synthesis in the forebrain might be inhibited. This is consistent with heightened arousal under stress as the proposed role of epinephrine at extrajunctional sites would be sedative/inhibitory. This is also consistent with the disproportionate depletion

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Fig 2. Hypothetical compartmentalization of synthesis and storage pools in bralnstem/medullary PNMT containing neurons. PNMT is found in the cytoplasm where its substrate, norepinephrine, can enter the synthetic pool when found in the cytoplasm. As norepinephrine is synthesized in the granule, which contains dopamine J3-hydroxylase (DI3H), entry into the cytoplasm occurs only upon reuptake of released norepinephrine or overflow from the storage granule. Epinephrine which is synthesized can be released from the cytoplasmic pool, or compete with norepinephrine or dopamine for uptake into the storage granule where it can act as cotransmitter. TH = tyrosine hydroxylase, AADC = aromatic amino acid decarboxylase.

Epinephrine in brain

of epinephrine during stress, and slow recovery of tissue content following this stress.

379

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norepinephrine could also act as an agonist at these same receptors, a metabolic or diffusional barrier must exist. In passing through this barrier, epinephrine, a somewhat more potent alpha-2 agonist may be formed. The evidence for this pool comes from several observations. Measurement of epinephrine in extracellular fluid shows it to be present in concentrations disproportionate with the tissue content and turnover (Routledge and Marsden, 1987a, b ). Rapid and marked accumulation of epinephrine is observed following medial forebraln bundle stimulation (Torda, 1977). Marked efflux of epinephrine, but not norepinephrine, is observed in portal blood following metabolic challenge or physiologic challenge (Gibbs, 1985, Johnston et al., 1984). Finally, our experiments with PNMT inhibitors demonstrate them to show rapid behavioral effects, inconsistent with the time course for depletion of tissue stores (Mefford, 1987b, Mefford et al., submitted a,b). It seems unlikely, based simply on the relative concentrations of epinephrine and norepinephrine found in most mammalian brains, that epinephrine co-stored in noradrenergic terminals has a unique function. This would represent the largest and most easily measured pool. It is likely that norepinephrine is released in large excess in the synaptic area and consequently the receptors in the synapse "see" a large excess of norepinephrine. The receptor affinities are such that it would be of little consequence which receptor subtype was present as a 10 to 20-fold excess of norepinephrine would preclude epinephrine being the primary agonist at these sites in most mammalian brains. The third pool, representing perhaps true neuronal epinephrine which might serve as a classical transmitter, is found primarily in the brainstem/medulla. The neurons from which this pool arises have been described in detail (see Hokfelt et al., 1984 for a comprehensive review) and represent a rather diffuse network with abundant local projections. The immunohistochemical evidence for the existence of this population of neurons containing immunoreactive PNMT is quite substantial. Pharmacological and surgical data suggests that this population differs from the PNMT-containing cells in the forebrain. If, as is the case with the bulk of brain PNMT (Ciaranello et al., 1969), the PNMT in these neurons is found in the cytoplasmic fraction and dopamine-13-hydroxylase in the granule, then synthesis of epinephrine in these neurons will be dependent upon the concentration of norepinephrine in the cytoplasm. This will be dependent upon overflow of norepinephrine from granules or reuptake of released norepinephrine. As a consequence of this requirement, these neurons are likely to have varying proportions of epinephrine and norepinephrine in the storage granules, dependent upon the neuronal activity or PNMT activity. Further, the formation of epinephrine in the cytoplasm allows for the possibility of release from a cytoplasmic pool, which could have a very short half life. Figure 2 shows the hypothetical PNMT-containing neuron in the brainstern/medulla. Based on the localization of these cell bodies and ascending projections it seems probable that these represent neurons with primitive function. Numerous investigators have connected the neurons of the A1 and A2 groups with regulation of blood pressure (Saavedra et al., 1976, Chalmers et al., 1979, Beart et al., 1979, Van den Buuse et al., 1984). Recent data from very careful retrograde tracing has demonstrated the afferents to the locus coeruleus to arise from these same cell body areas (Aston-Jones et al., 1986). Aston-Jones and coworkers (Pieribone et al. 1987) recently demonstrated that PNMT containing ceils provide 20-30% of the innervation of the locus coeruleus, suggesting a function in providing tonic inhibitory regulation of sensory information. Such

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connections would be consistent with a general function in regulation of arousal. When these neurons contain primarily norepinephrine preferential synaptic stimulation of beta-1 and/or alpha-1 receptors and excitation or arousal might occur. When these neurons contain primarily epinephrine as could gradually become the case after repeated firing, preferential stimulation of alpha-2 receptors and sedation or inhibition would occur. Release from a cytoplasmic pool, modified by reuptake of released norepinephrine, might serve the same function, or have a more general inhibitory action as this release might not be synaptic and would allow the synthesizing cell to influence numerous associated ceils. This could provide a mechanism for habituation to repeated sensory stimuli for the diurnal cycle in arousal as has previously been suggested (Fuxe et al., 1974). It is proposed then, that these brainstem "epinephrine" neurons act to regulate the threshold for stimulation of the locus coeruleus either directly via alpha-2 receptors at the somatodendritic level or via inhibitor influence on excitatory projections to the locus coeruleus.

In this way

epinephrine-synthesizing neurons in the brainstem could provide tonic regulation of sensory input to the locus coeruleus (Mefford and Potter, 1988). This view is consistent with the observations of epinephrine content and general arousal among various mananalian species. Making measurements which differentiate these various pools is quite difficult. Most measurements of tissue content probably reflect primarily the least significant pool, co-stored in noradrenergic terminals. Some evidence exists for modest differences in the N-methyltransferase enzymes in the brainstem compared to the hypothalamus (Pendleton et al., 1977a). This suggests a means of differentiating the neuronal PNMT from PNMT in other types of cells or different forms of PNMT. Equilibrium dialysis appears to be the method of choice for measuring the functional, hormonal pool. These three measurements; changes in brainstem PNMT activity, which might reflect neuronal PNMT and therefore "epinephrine neurons"; tissue content, reflecting primarily epinephrine co-stored in noradrenergic terminals and; extracelluiar epinephrine, reflecting the hormonal pool, may give insight into the functional significance of these three systems.

3. Significance and Conclusions The data presented and reviewed demonstrate epinephrine to be found in brain tissue throughout phylogeny. Great variation exists between the various species and within each class. Epinephrine becomes a significant contributor to the total catecholamine content of brain in the amphibians and reptiles, while appearing to be a minor constituent in the fishes which have been examined. Mammals have been most extensively studied, and show considerable variation. Carnivores appear to have relatively high epinephrine content while herbivores appear to have little or none. Omnivores fall somewhere between these two extremes. Epinephrine and PNMT are widely distributed in primate brain. Highest epinephrine content is found in the hypothalamus, midline structures and brainstem, maintaining this relative distribution throughout phylogeny. Higher functioning cortical areas contain low concentrations of epinephrine but do generally contain significant PNMT activity. The ontogeny of epinephrine in rat brain appears to generally parallel phylogeny, with the relative presence of epinephrine to be decreased as activation is increased. At least in rat forebrain, epinephrine appears to be found primarily co-stored in noradrenergic nerve endings, while synthesized in an extraneuronal compartment, perhaps gila or a neuron dissociated from the storage site. Alternatively, both storage and synthesis could occur in a non-neuronal secretory type cell,

Epinephrine in brain

perhaps similar to chromaff'm cells, not containing the full complement of biosynthetic enzymes. In the brainstem, neurons are present which contain the full complement of biosynthetic enzymes. Thus, these neurons may form epinephrine as the endprodnct catechol~mlne, release epinephrine from storage granules and may be true epinephrine neurons. Formed in the cytoplasm of these neurons, it is possible that epinephrine might also be released from a cytoplasmic pool. Formed in an extraneuronal compartment in the forebrain, epinephrine may act as a hormone, responding to the release of norepinephrine from projections of the A1 and A2 noradrenergic cell groups. This pool should show slow increases since change is dependent upon synthesis, but rapid depletion as no storage pool may present in these extraneuronal sites. The functional significance of epinephrine in brain is at best questionable, however, several lines of evidence suggest that under resting conditions, extracellular epinephrine is equal to norepinephrine. This being the case, epinephrine would be the primary agonist at nonsynaptic alpha-2 and beta-2 receptors exposed to this pool. The role of epinephrine as agonist at these receptors would be tonic in nature and likely inhibitory as evidenced by the effects of alpha-2 agonists. Administration of inhibitors of PNMT suggest involvement of epinephrine synthesis in reward mechanisms (Katz and Carroll, 1977, Katz et al., 1978a,b), motor activity (Katz et al. 1978, 1979) as well as sedation and intoxication (Mefford et al., 1987). In each of these cases, the observed effects may be secondary to release of norepinephrine. Several neuroendoerine functions have been suggested for epinephrine including stimulation of growth hormone release (Gold et al., 1978), inhibition of ACTH and corticosterone release (Roth et al., 1981, Mezey et al., 1985) and regulation of thyrotropin releasing hormone (Klibanski et al., 1983). The available literature suggests that epinephrine in brain serves quite the opposite function of epinephrine released from the adrenal medulla. Cannon, nearly seventy years ago (Cannon, 1922) suggested that adrenal epinephrine had an "emergency" function. This function, as evidenced in the effects of peripherally administered epinephrine, is generally excitatory, increasing heart rate and stroke volume, breathing rate and inducing several peripheral signs of anxiety (Rothballer, 1959). Epinephrine in the brain appears to serve a tonic inhibitory role (Mandell and Spooner, 1968). Because its presence in extracellular space should be dependent upon release of norepinephrine and its subsequent uptake and synthesis, rapid, acute effects are likely to be slight. The hypothesis that catecholamines could be synthesized and released from non-neuronal cells in the brain was proposed nearly fifteen years ago (Shoemaker, 1973), and probable secretory cells in the periventricular hypothalamus of the frog have been described (McKenna and Rosenbluth, 1971). Forebrain epinephrine synthesis in mammals may be a prime example of this type of secretory process. It remains to be seen whether epinephrine synthesized in the brain can reach the periphery and have any hormonal effects outside the CNS, however, yon Euler (von Euler et al., 1961) demonstrated the slow time course formation and excretion of epinephrine in adrenaleetomized subjects to administered insulin, suggesting this possibility. Epinephrine is the least studied of the catecholamines in the brain. Perhaps this is appropriate as its role as a neuromodulator may be quite minor or even nonexistent. It is also possible that, as noted by Carlsson (1976) and Fuller (1982), the brain does not function as a democracy and the low tissue content of epinephrine belies its significance.

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Acknowledgements This review is the product of many fruitful discussions particularly with Drs. Gosta Jonsson and Tomas Hokfelt of the Karolinska Institute, Dr. Ralph Adams, The University of Kansas, Dr. Kevin Roth, Washington University Medical School and Dr. Ray Fuller, Eli Lilly Company. I am particularly indebted to my former students at Boston College, Mary M. Cooney, Catherine Swible-Keane, Tracy Durkin and Ed Caliguri whose efforts provided a good deal of insight on epinephrine in brain.

Re£erenees Abel, J.J. (1902) Method of preparing epinephrine and its compounds, Bull. J. HoD. Hosp, 13, 29-35. Aston-Jones, G., Ennis, M., Pieribone, V.A., Nickell, W.T. and Shipley, M.T. (1986) The brain nucleus locus coeruleus: Restricted afferent control of a broad efferent network, Science, 234, 734-737. Barchas, J.D., Ciaranello, R.D. and Steinman, A.M. (1969) Epinephrine formation and metabolism in mammalian brain, Biol, Psychiat., 1, 31-48. Beart, P.M. (1979) Adrenaline. The cryptic central eatecholamine, Trends Neurol. Sci., 2, 295-297. Beart, P.M., Prosser, D. and Louis, W.J. (1979) Adrenaline and phenylethanolamineN-methyltransferase in rat medullary and anterior hypothalamic preoptie nuclei, I. Neurochem., 33, 947-950. Berger, P.A., King, R., Lemoine, P., Mefford, I.N. and Barchas, J.D. (1984) Cerebrospinal fluid epinephrine concentrations--Discrimination of subtypes of depression and schizophrenia, P~vchooharmacolot, v Bulletin, 20, 412-415. Blaschko, H. (1942) T-lieactivity of l(-)dopa decarboxylase, ~I.Physiol., 101,337-345. Bohn, M.C., Goldstein, M. and Black, I.B. (1986) Expression and development of phenylethanolamine N-methyltransferase (PNMT) in rat-brain stem. Studies with glucocorticoids, Developmental BioloCy, 114(1), 180-193. Brownstein, M.J., Palkovits, M., Tappaz, M.L., Saavedra, J.M. and Kizer, J.S. (1976) Effect of surgical isolation of the hypothaiarnus on its neurotransmitter content, Brain Res., 117,287-295. Caliguri, E.J. (1986) Ph.D. Thesis, Department of Chemistry, Boston College, Chestnut Hill, Massachusetts. Cannon, W.B. (1922) Emergency function of the medulla, Endocrinol. and Metab.. 2, 171-180. Carlsson, A. (1959) The occurrence, distribution and physiological role of cateeholamines in the nervous system, Pharmacological Reviews, 11,490-493. Carlsson, A. (1976) Centrai catecholamines. Adv. Clin. Pharmacol. 12, 1-4. Cedarbaum J.M. and Aghajanian, G.K. (1976) Noradrenergic neurons of the locus coeruleus: inhibition by epinephrine and activation by the antagonist piperoxane, Brain Res.. 112, 412-419. Chalmers, J.P., Petty, M.A. and Reid, J.L. (1979) Participation of adrenergic and noradrenergic neurones in central connections of arterial baroreceptor reflexes in the rat, Circulation Research, 45, 516-522 Chamba, G. and Renaud, B. (1983) Distribution of tyrosine hydroxylase, dopamine-~-hydroxylase and phenylethanolamine-N-methyltransferase activities in coronal sections of the rat lower brainstem, Brain Reg., 259, 95-102. Christensen, N.L, Vestergaard, P., Sorensen, T. and Rafaelson, O.J. (1980) Cerebrospinal fluid noradrenaline and adrenaline in depressed patients, Acta Psychiat. Scand. 56, 178-182. Ciaranello, R.D. (1978) Regulation of phenylethanolamine-N-methyltransferase synthesis and degradation. 1. Regulation by rat adrenal glucocorticoids., Molecular Pharmacol.. 14, 478-489. Ciuranello, R.D., Barchas, R.E., Byers, G.S., Stemmle, D.W. and Barchas, J.D. (1969) Enzymatic synthesis of adrenaline in mammalian brain, Nature (London), 221,368-369. Cooney. M.M., Conaway, D.H. and Mefford, I.N. (1985) Epinephrine, norepinephrine and dopamine concentrations in amphibian brain, Como. Biochem. Physiol., 82C, 395-397. Cooney, M.M. (1986) Master's Thesis, Department of Chemistry, Boston College, Chestnut Hill, Massachusetts. Drew, G.M., Gower, A.J., and Marriott, A.S. (1979) or-2 Adrenoreceptors mediate clonidine-induced sedation in the rat., Br. J. Pharmacol., 67, 133-141.

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