Strategies in Neuroendocrine Neurophysiology. Institute of Animal Behavior, Rutgers University, Newark, New Jersey 07102

Transcription

1 AM. ZOOLOCIST, 11: (1971) Strateges n Neuroendocrne Neurophysology BARRY R. KOMISARUK Insttute of Anmal Behavor, Rutgers Unversty, Newark, New Jersey SYNOPSIS. Hormonal effects on selected ndcators of neural actvty are revewed wth reference to neuroendocrne mechansms. The nature of bran recordngs and ther advantages and dsadvantages n neuroendocrne applcatons are dscussed. The problems of localzaton of neuroendocrne mechansms n place and tme are presented, wth ndcatons of whch processes may be best suted to neurophysologcal analyss. INTRODUCTION In neuroedocrne processes, the ptutarytarget organ mechansms respond to the nternal and external envronment va the central nervous system, and the resultng change n hormone secreton feeds back and changes the actvty of ths system at multple levels n the bran. Thus, the output of the bran-ptutary and branbehavoral systems s regulated. The followng are some examples of ths effect. Sawyer and Markee (1959) reported that whle a small proporton of naturally estrous rabbts ovulate reflexly to vagnal stmulaton, estrogen admnstraton markedly ncreased the proporton ovulatng n response to vagnal stmulaton. Treatment wth neurotransmtter blockng agents abolshed ths response, further ndcatng that t was neurally medated. Spontaneous ovulaton was absent n anestrous rabbts, but after estrogen treatment, a small proporton ovulated even wthout stmulaton. Thus, unless estrogen senstvty of the ptutary s a controllng factor, the ovulaton reflex s apparently an estrogen-senstve mechansm whose actvaton threshold can occasonally be bypassed wth estrogen treat- Contrbuton No. 113 from the Insttute of Anmal Behavor. Ths study was supported by Research Grant MH and Research Scentst Development Award 1-K2-MH-14, from the Natonal Insttute of Mental Health. It has receved bblographc ad from the Unversty of Calforna, Los Angeles Bran Informaton Servce, whch s a part of the Natonal Informaton Network of the Natonal Insttute of Neurologcal Dseases and Blndness and supported under contract PH menl alone. In the rat, whch normally ovulates n the absence of cotus, ovulaton can nevertheless be nduced n response to cotus or vagnal stmulaton under varous condtons n whch spontaneous ovulaton does not occur (Dempsey and Searles, 1943; Aron et al., 1966; Everett, 1967; Zarrow and Clark, 1968). Everett (1964) elcted ovulaton n rats by electrochemcal stmulaton n and near the medal preoptc regon, and found that the threshold ovulatonnducng current durng proestrus was halt that durng destrus. We found (Ramrez et al., 1967) that whle cervcal stmulaton n urethane-anesthetzed rats nduced a sleeplke Electroencephalogram (EEG) pattern to a varable extent throughout the estrous cycle, vrtually all the rats showed relable "sleep" nducton when they were n late destrus or early proestrus. There s other dverse evdence that hormones may act largely by changng the actvty level of already functonal neural mechansms. For example, cervcal stmulaton appears to be so potent a stmulus for elctng the lordoss reflex n rats that the reflex response can be elcted n the absence of ovaran and adrenal sterods. Small dosages of estrogen ncrease the ntensty of the reflex even though estrogen s apparently not essental for the operaton of the reflex (Komsaruk, 1972) (Fgs. 1, 2). Thus, n nvestgatng the effects of hormones on the bran, one must realze that the hormone may act largely quanttatvely on exstng mechansms; the latter are themselves lkely to be connected wth a varety of bran systems {see Sawyer, 741

2 742 BARRY R. KOMISARUR CHARACTERISTICS OF BRAIN ACTIVITY It may frst be helpful to dscuss the several types of bran recordng methods avalable and what they represent. Sngleor mult-unt recordngs ndcate whether neurons n the very close vcnty of the electrode are generatng acton potentals. The same electrodes also record slower, feld-potental fluctuatons concurrently, and f these are not fltered out electroncally, the acton potentals are seen to "rde" upon the slow-potental undulatons. The latter represent the EEG, and there s substantal evdence that dscrete bursts of acton potentals can be related to ndvdual waves of the EEG (Fg. 3). Accordng to Morrell (1967), "The consensus s that the EEG s the algebrac sum of hundreds of thousands of synaptc potentals n the many thousands of neurons FIG. 1. Inducton of lordoss by probng the vag- whch le wthn the 'feld' of a gross renal cervx n conjuncton wth flank-perneum cordng electrode." The sze of the feld s palpaton. The ntensty of the reflex response was measured as dfference n mllmeters be- related to the surface area of the recordtween heghts of tp of nose and low pont of ng electrodes. Snce these synaptc potenback plus that between top of rump and low tals can depolarze post-synaptc neurons, pont of back. (N:nose; B:back; R:rump). B. thereby nducng the generaton of acton Lordoss n ovarectomzed, untreated rat. A. No lordoss n a dfferent, smlarly treated, rat. The potentals, the two types of recordngs are functonally related. bass for ths dfference s not known. C. Intense lordoss n a rat whch receved a sngle njecton There are several dstnct advantages of of 10 ng Estradol Benzoate (EB)/100 g bw about lomdosis HCKjM- 41 hours prevously. Group data are presented n Fgure 2. D. Lordoss n an androgenzed female. F. Lordoss to probng of the rectum n an ntact male whch receved 10 /»g EB/100 g bw?bou 42 hours prevously. E. No lordoss n an untreated ntact male. No untreated males showed lordoss. These fndngs ndcate that estrogen facltates the lordoss reflex, but that at least n females, ovaran estrogen s not essental for ths response. (From Komsaruk, 1971.) 1967, for revew), and thus are subject to non-hormonal controllng mechansms. Snce hormones also exert marked but dffuse effects on bran exctablty (Woolley FIG. 2. Lordoss heght response to graded doses and Tmras, 1962; Heuser et al., 1965; of EB admnstered hours before testng n ovarectomzed rats (on rght). Numbers above Marcus et al., 1966; Woodbury and Vern- abscssa are numbers of anmals per group. Sold adaks, 1966, 1967), the problem of dent- bars on left ndcate lordoss heghts n ntact fyng "specfc" hormonal effects on bran females at selected phases of the estrous cycle, mechansms s complex. I shall attempt to ndcatng strongest responses n vagnal proestrus and n the transton from destrus to proestrus, revew the nature and sgnfcance of some and lowest responses n early destrus, metestrus, of these effects n terms of hormonal acton and estrus, smlar to that $ weeks after ovaron bran actvty. cctomy (center bar). (From Komsaruk, 1972.) p p t E M O DP OVX ESTRAOIOL BENZOATE^ 3 /l00 3 BW

3 NEUROENDOCRINE MECHANISMS 743 UPC UNITS MB UNITS FLAXEDIL IMMOBILIZATION (D-F) HPC UNITS FIG. 3. Synchrony between bursts of neuronal actvty and ndvdual EEG waves. The hppocampal (HPC) unt and EEG actvty was taken from the same electrode and fltered dfferentally then dsplayed smultaneously. Note that correlatons seen n unanesthetzed rat (A and B) persst after t s mmoblzed wth Flaxedl (D-F). No clear correlaton s seen n the mamllary body record (MB). Chewng artfacts are shown n C for comparson wth neuronal actvty. Calbratons: 1 second and 100 ^V. (From Komsaruk, 1970.) recordng acton potentals (. e., sngle- or mult-unt recordngs) rather than (or n addton to) EEG: (1) the number and tmng of acton potentals can be measured readly; (2) the level of exctaton can be quantfed; and (3) nhbton of neuronal actvty can be dentfed. These measures provde nformaton as to whether the regon beng recorded from can be transmttng actvty to dstant regons. In contrast, (1) the EEG can exst n the absence of acton potentals, snce a partcular pattern of synaptc potentals need not generate acton potentals; (2) moment-to-moment nhbton of actvty can not be dentfed n the EEG; and (3) there s, at best, restrcted sgnfcance n quantfyng the number of EEG waves per unt tme or ther ampltude. Furthermore, for a gven electrode, snce small lesons made through recordng electrodes abolshed acton potentals but not the EEG, the EEG feld s less localzed than the acton potental feld (Rudomn et ah, 1965) although some localzaton can be obtaned (Morrell, 1967). The major change whch occurs n the EEG s one from hgh ampltude slow waves (sleep-lke condtons) to relatvely low ampltude fast waves (arousal). The terms "synchronous" and "desynchronous" appled to these patterns, respectvely, refer to the degree of smultanety of the synaptc potentals (Morrell, 1967), possbly reflectng the fluctuatng degree of functonal dfferentaton of the neurons n the recordng feld (Lndsley, 1961). Evoked potentals whch are perturbatons n the EEG are termed "slow-wave evoked potentals;" perturbatons of neuronal acton potentals are termed "evoked unt dscharges." These are conducted to the recordng ste along afferent channels from a dstant stmulaton ste (Landau, 1967), and snce both types of recordngs often show concomtant changes (Fox and O'Bren, 1965), they also probably represent two aspects of the same basc neural process. Ths relatvely fast bran actvty (over 1 cycle per second) "rdes" upon, and s modulated by, much slower bran ac-

4 744 BARRY R. KOMISARUK J FIG. 4. Schematc dagram of the relatonshp between neuronal, EEG, and steady-potental electrcal recordngs of bran actvty. The neuronal (unt) spkes "rde" on the EEG and can be fltered dfferentally, as n Fgure 3. Ths actvty "rdes" on the steady potental shfts seen n the left porton of the fgure. Calbratons are approxmate. tvty the "steady potental" ("DC shft") whch fluctuates wth perods of many seconds (Kawamura and Sawyer, 1964; Rowland, 1967; Fromm and Bond, 1967). These relatonshps are represented dagrammatcally n Fgure 4. Recordng these electrcal sgns of bran actvty has provded nformaton on the stes, latences, and nature of hormonal effects upon the central nervous system, and has shown also that hormones modulate the responsveness of neural systems to envronmental stmul. HORMONAL EFFECTS ON BRAIN ACTIVITY EEG Bran recordng technques have been used to dentfy the tme, place and nature of neuroendocrne events (for recent revews, see Beyer and Sawyer, 1969; Cross and Slver, 1966; Cross, 1964). Porter et al. (1957) observed that EEG hgh ampltude slow waves were nduced n the hypothalamus, n and around the medan forebran bundle, durng vagnal stmulaton n estrous, but not anestrous, cats. Snce vagnal stmulaton nduces ovulaton n estrous but not anestrous cats, they suggested that ths actvty s related to reflex ovulaton. Recently, however, Sutn and Mchael (1970) elcted smlar EEG actvty n estrous, as well as anestrous cats, shortly after vagnal probng. Smlar actvty was also seen n response to tal pnch. They thus concluded that ths pattern s not stmulus-specfc and s not related to the hormonal or behavoral state. Ths dscrepancy llustrates one of the major confoundng factors n bran recordng, that s, tryng to separate changes n actvty whch represent generalzed effects from those more specfc to the mechansm of nterest. A smlar problem was encountered when Barraclough (1960) reported that n rats under pentobarbtal anesthesa, hgh ampltude EEG waves could be nduced n the hypothalamus by probng the vagnal cervx n proestrus or estrus, but not n destrus or after ovarectomy; Margherta et al. (1965), however, observed that ths type of actvty could be nduced n all stages of the estrous cycle by vagnal as well as any other arousng stmul and was related to the level of anesthesa. Perhaps one of the dffcultes n these studes s that hormones change the general exctablty level of the bran, and such nonspecfc changes may normally affect a varety of specfc mechansms, so that the change observed under varous hormonal condtons may be largely quanttatve rather than qualtatve. Kawakam and Sawyer (1959a) showed that estrogen and progesterone exert profound effects upon the arousablty of rabbts. These fndngs have been confrmed by Endrocz (1967). The mnmum voltage requred to arouse the bran (.e., produce a desynchronzed EEG n response to electrcal stmulaton of the mdbran retcular formaton) from spontaneous sleep decreased gradually for several hours after a subcutaneous njecton of progesterone n estrogen-prmed rabbts (the decrease was faster when progesterone was njected ntraventrcularly). The arousal threshold 24 hours later was markedly elevated. Elevaton of ths "arousal threshold" was assocated wth refusal to mate. The functonal relatonshp between behavoral sexual receptvty and arousal threshold whch s measured durng sleep s not known, but the non-specfc exctablty of the bran s strongly nfluenced by progesterone n a bphasc effect. When varous components of the lmbc system or 4 I

5 NEUROENDOCRINE MECHANISMS 745 the ventromedal hypothalamus were stmulated electrcally, an "EEG after-reacton" developed whch s now recognzed as paradoxcal sleep (Sawyer, 1966). Ths s characterzed by a desynchronzed EEG n the cortex and a hypersynchronzed, 8 cycles per second, theta pattern n the lmbc system and hypothalamus. It resembles the EEG durng wakefulness, but the anmal s relatvely resstant to beng awakened and deeply asleep (thus the term "paradoxcal" sleep). The threshold voltage requred to elct ths sleep pattern followed a smlar but not dentcal tme course to the arousal threshold. Dfferental effects on the two thresholds were observed wth certan progestatonal contraceptve drugs, such as norethynodrel (Sawyer and Kawakam, 1961). These rabbts mated, but ovulaton faled to occur. A smlar type of effect was obtaned wth hgh dosages of estradol benzoate or testosterone proponate (Sawyer and Kawakam, 1961; Kawakam and Sawyer, 1967). Snce the "EEG afterreacton threshold" was elevated but the "arousal threshold" was not elevated, they hypotheszed that the arousal threshold was related more closely to the sexual behavor, and the EEG afterreacton threshold was more closely related to ovulaton. Supportng evdence was obtaned wth long-actng progestatonal agents, such as Delalutn, whch prolonged the nterval of sexual receptvty and ablty to ovulate, and concomtantly delayed the elevaton of the two thresholds. Ths paradoxcal sleep pattern occurs soon after copulaton or vagnal stmulaton (Sawyer and Kawakam, 1959), but snce t occurs after the ntaton of ovulatng hormone (LH) release, t s apparently not essental for ptutary actvaton (Beyer and Sawyer, 1969). It may, however, be related to a postve feedback mechansm, for LH and certan other ptutary hormones nduce ths EEG pattern (Kawakam and Sawyer, 19596), and t seldom occurs n hypophysectomzed rabbts (Spes and Sawyer, see Beyer and Sawyer, 1969). In rats, ntravenous admnstraton of LH (Ramrez et ah, 1967; Terasawa et ah, 1969) or ACTH (Sawyer et ah, 1968) changes actvty n the hypothalamus wthn mnutes. Further evdence that paradoxcal sleep may n some way be related to the ovulaton process s that there was 2.5 tmes more paradoxcal sleep after copulaton than precedng t n rabbts whch ovulated as a result of ths copulaton. Moreover, n rabbts whch faled to ovulate there was only 1.2 tmes more paradoxcal sleep after copulaton than before, n a comparable 6 hour perod (Kawakam, 1966). It s curous that the amount of deep sleep ncreases at a tme when the arousablty of the rabbts also ncreases (.e., whle the arousal threshold decreases). Steady-potental shfts have been observed n rats (Law and Sackett, 1965) and cats (Rowland, 1967) upon vagnal stmulaton. Evoked Potentals Hormones have been shown to affect both the speed of conducton and the ampltudes of evoked slow-wave potentals (Oshma and Gorbman, 1969). Thyroxne accelerates, and cortsol retards, the ontogeny of sensory evoked potentals n the cortex of mmature rats (Salas and Schapro, 1970). The speed of conducton from the mdbran retcular formaton to the cerebral cortex n the cat was slowed by adrenalectomy, accelerated by adrenal cortcods, and accelerated stll more by dexamethasone, a "super glucocortcod" (Chambers et ah, 1963). The ampltudes of spnal segmental mono- and polysynaptc potentals n cats were ncreased wthn 1.5 hours after systemc estrogen njecton; progesterone alone depressed the ampltude, but had synergstc effects n combnaton wth estrogen (Kawakam, 1960). Kawakam and Terasawa (1967) found that exogenous estrogen ncreased the ampltude of the late component of the evoked potental from scatc nerve or sacral root stmulaton to the mdbran retcular formaton or arcuate nucleus, but t dd not affect the ampltude of early responses or lemnscal responses. They nterpreted ths to mean that "an

6 746 BARRY R. KOMISARUK FIG. 5. Tracngs of evoked slow-wave potentals recorded n the basal hypothalamus (premamllary area) n response to sngle-pulse electrcal stmulaton of the mdbran retcular formaton. The actvty changes dramatcally n relaton to the behavor of the rabbt at the tme each record was taken. Each of the four records s the "average" of 100 stmul, gven 1 second apart, and processed by a Computer of Average Transents. The lordoss record was obtaned durng perneal tappng, 15 mnutes after the alert record (durng whch the rabbt was restraned smlarly but wthout perneal tappng). Note that ths dfference was ntermedate between the responses durng slow wave spndle sleep and paradoxcal sleep. (Komsaruk and Sawyer, unpublshed.) ncrease n the number of synaptc passages seems to amplfy the exctatory or nhbtory changes smlar n manner to those n the retcular multsynaptc system." Ths s remnscent of a smlar effect observed by French et al. (1953), who found that when evoked potentals were recorded n the tegmentum and sensory cortex by stmulaton of the scatc nerve, ntravenous pentobarbtal admnstraton had a much greater depressant effect on the tegmental (multsynaptc) pathway than the cortcal (paucsynaptc) pathway. Endrocz (1969) reported dfferental effects of estrogen and progesterone on evoked slow-wave potentals n the bran stem and hypothalamus n rats, some changes occurng wthn 5-10 mnutes of an ntravenous (v) njecton of progesteone. Whle these may be relable effects, t must be recognzed that evoked potentals may be as lable as the EEG's of whch they are a part, and that non-hormonally dependent changes n general bran or behavoral actvty may exert profound effects on the evoked potental. Ths s especally sgnfcant when the mdbran retcular formaton s nvolved. For example, Fgure 5 shows that n a stable hormonal condton (all records taken wthn 90 mnutes of each other) there are marked changes n the evoked potentals related to what the rabbt was dong when the evoked potentals were obtaned. It s, therefore, of utmost mportance to montor ndces of bran actvty other than only the one of nterest. The selecton and measurement of approprate controls s crtcal, for nonhormonal varables may contrbute more to changng the evoked potental than does the hormone under study. Neuronal Actvty By recordng sngle- or multunt actvty rather than EEG or slow-wave evoked potentals, t s possble to specfy wth greater precson the locaton, tme, and perhaps nature of neural actvty modfcatons whch are nduced by hormones. The actvty of a very large proporton of neurons as well as the EEG s, however, strongly nfluenced by non-specfc arousal of the bran (ndcated by whether changes n ther actvty are correlated wth synchronzed or desynchronzed changes n the cortcal EEG). Hypothalamc and preoptc area neurons are more ndependent of EEG changes than thalamc, cortcal, or mdbran retcular formaton neurons (Komsaruk et al., 1967; Lncoln, 1969a; Kawakam et al., 1970; Terasawa and Sawyer, 1970). Haller and Barraclough (1970) found that n terms of frng patterns, ventromedal hypothalamc neurons were actually more closely related to cortcal EEG changes than were thalamc neurons. Even hypothalamc neurons whch respond n hghly dscrete patterns to sensory stmulaton are concurrently modulated by the general actvty le\el of the bran (Fg. 6) (Komsaruk and Beyer, 1972). Snce the actvty of at least 4 1 j

7 NEUROENDOCRINE MECHANISMS 747 t A-DURING SLEEPLIKE EEG C- DURING SLEEPLIKE EEG Loterol Hypothalomus (23) B-DURING AROUSED (THETA)EEG Lateral Hypothalamus (16) D- DURING AROUSED (THETA)EEG F.- DURING XYLENE PRESENTATION FIG. 6. Influence of non-specfc arousal on hypothalamc neuronal responses to olfactory bulb stmulaton. Short-latency, relatvely dscrete, responses are clearly facltated durng aroused perods n rats under urclhanc anesthesa (contrast B and D wth A and C, respectvely). Other modulatng factors are seen n E and F. Each horzontal lne s a sngle tral; stmulus artfacts form a merged vertcal lne toward the left of each fgure. Stmulus ntensty was held constant. Calbraton: 10 msec. (Komsartk and Beyer, 1972.) some hypothalamc neurons s nfluenced by both olfactory and non-specfc arousal stmul, f hormones are shown to change the actvty of hypothalamc neurons, t may be because the hormones have affected one of the nput mechansms to these neurons, such as the mdbran. retcular formaton. In other words, fndng a hormone-related change n actvty n a hypothalamc neuron does not necessarly mean that the hormone s actng drectly upon that neuron. The relatonshp between sngle unt actvty and non-specfc bran exctablty was not taken nto account when Barraclough and Cross (1963) showed that wthn several mnutes, v admnstraton of progesterone n propylene glycol nhbted the response of hypothalamc neurons to stmulaton of the vagnal cervx, but not to pnch or cold stmul. We repeated and confrmed ther fndngs but also observed that actvaton of these neurons could be elcted by any stmul whch also produced an EEG arousal (pnch, vagnal, or rectal stmulaton) but not by stmul whch dd not produce arousal (touch, lght, warm water) (Komsaruk et al., 1967). Progesterone n propylene glycol nduced a sleeplke EEG, and then each of these stmul became markedly less effectve n actvatng the unts and arousng the EEG. Durng ths phase, vagnal cervx stmulaton was relatvely weak n ts ablty to arouse the EEG and actvate the neurons, but pnch and other strong stmul could stll do so, although the ntensty and duraton of ther effect was reduced. Recovey to ntal characterstcs occurred after about 1 hour. Even n the context of a dffuse response to a hormone, dfferental responses among neurons were observed (Fg. 7). All the unts whch we found to be unaffected by progesterone were also unaffected durng spontaneous or nduced EEG changes. Lncoln (19696) found some neurons n the anteror hypothalamus that were actvated by cervcal stmulaton ndependently of EEG changes; however, such neurons were also unaffected by progesterone (Lncoln, 1969c). Inducton of sleeplke EEG actvty by progesterone was also observed by Kobayash el al. (1962), Ara et al. (1967), and Ramrez et al. (1967). Heuser el al. (1967) nduced behavoral as well as EEG sleep by ntroducng progesterone n lqud or crystallne form drectly nto the preoptc area of cats, and Komsaruk (1967) n doves nduced ncubaton (a quescent behavoral state) and suppressed courtshp (an androgen-dependent behavor pattern) by mplantaton of crystallne progesterone nto the preoptc area and other forebran regons. The problem of solvent or vehcle selecton s of consderable mportance, especally when attemptng to analyze rapd (and possbly generalzed) hormone effects. Whle we (Komsaruk et al., 1967) found sgnfcantly greater depresson of neuronal responsveness wth progesterone n propylene glycol than wth propylene glycol

8 748 BARRY R. KOMISARUK 20 mn after 20-OH 50 mn after 20~0H UNIT I I I II I I III I I ' " I 1 1IIII I Illl " m HI [ 11 mm, H, n CORTICAL INTEG. MULTI- UNIT 50mV OB-FC EEG I 150/JV FIG. 7. Dfferental recovery from progestn (20 a - hydroxy-prcgn-4-en-3-oe) depresson. Twenty mnutes after IV njecton of the progestn n propylene glycol, the cortcal EEG shows a short spontaneous "arousal" whch s accompaned by an actvaton of a pool of cortcal neurons (ntegrated mult-unt record). The sngle cortcal neuron s alone, the vehcle alone had a smlar effect. However, Ara et al. (1967) obtaned a dose-dependent progesterone nducton of cortcal sleep-lke EEG actvty upon admnstraton of progesterone n propylene glycol v, and lttle f any effect of propylene glycol alone. The estrogen background of the rats was apparently antagonstc to progesterone, for progesterone (75 /tg) n spayed rats elcted substantally longer sleep-lke actvty than equal doses of progesterone n ntact, or spayedestrogen prmed, rats. Terasawa and Sawyer (1970) have shown that progesterone ( ng) njected subcutaneously n sesame ol nduced a sleep-lke EEG wthn one hour n estrogen-prmed rats. Lncoln (1969c) found that progesterone n propylene glycol, and propylene glycol alone, both produced equally depressve effects. However, progesterone mcrocrystals n salne (but not salne alone) njected ntravenously produced prolonged perods of synchronzed EEG, durng whch hypothalamc unts showed large changes n actvty. Thus, progesterone, ndependently of the vehcle, can apparently nfluence h\pothalamc and othe neuom, pehaps medated at least n part by non-specfc 15 sec unaffected at ths tme. However, 30 mnutes later, the spontaneous arousal of the EEG and the concomtant ncrease n actvty of the multunt pool s accompaned by an actvaton of ths sngle neuron. (Unpublshed observatons of Komsaruk, McDonald, Whtmoyer, and Sawyer.) arousal mechansms. Under approprate condtons, local neuroendocrne mechansms appear to exst, ndependent of non-specfc arousal. Pekary et al. (1967) faled to block ovulaton or alter target gland weghts wth mdbran-hypothalamc lesons, and electrcal stmulaton of ths regon dd not nduce ovulaton (Pekary el al., 1967; Kawakam et al., 1970). Relatvely local hormone mplantaton nto the hypothalamus alters a varety of ptutary and behavoral feedback mechansms (see revews by Sawyer et al., 1966; Lsk, 1967), and some degree of competence of control of ptutary secreton perssts n hypothalamc "slands" (Halasz and Gorsk, 1967; Halasz et ah, J967). Localzed nfluences of hormones n ths regon (.e., not affectng other regons) have been suggested by evdence from systemc hormone njecton (Ramrez et al., 1967; Sawyer et al., 1968; Terasawa et al., 1969). A dfferent approach has been to restrct the bran regon whch can be affected by the hormone. Thus, cortsol admnstered v decreases frng rates of neurons n hypothalamc "slands" (Feldnan and Same, 1970), although n ths t\pe of preparaton oxytocn s neffectve

9 NEUROENDOCRINE MECHANISMS 749 L (Cross and Dyer, 1969). Furthermore, dexamethasone and ACTH ntroduced mcroelectrophoretcally nto ths regon through multbarrellcd mcroelectrodes change (manly decrease and ncrease, respectvely) the frng actvty of sngle neurons (Stener et al., 1969; Ruf and Stener, 1967). Cortcosterone mcronjectons nto the hppocampus changed the actvty of pyramdal neurons (Pfaff, personal communcaton), and testosterone proponate mcronjectons nto the preoptc area changed the responsveness of prcoptc neurons to olfactory bulb stmulaton wthn 5-15 mnutes (Pfaff and Pfaffmann, 1969). CORRELATING NEURAL ACTIVITY WITH NEUROENDOCRINE EVENTS: TEMPORAL FACTORS Hormonal nfluences on bran actvty have been analyzed by usng bascally two approaches. One s to compare bran actvty or responsveness for a short samplng perod n treated, n contrast to untreated anmals, thus generatng two populatons, only one of whch s under the nfluence of a hormone treatment (Cross and Slver, 1965; Lncoln and Cross, 1967; Lncoln, 1967; Dafny and Feldman, 1970). Ths assumes that equvalent bran regons and neuronal populatons are beng observed n the two samplng populatons. Wth ths approach, two dfferent bran regons may be compared under two dfferent hormone condtons for determnng regonal dfferences n response to hormones (Alcaraz et al., 1969, found dfferences n hypothalamc vs. mdbran retcular formaton neuronal responsveness n ovarectomzed vs. estrogen-treated cats). The second approach s to record at a gven ste the actvty or responsveness before, n contrast to after, the hormone treatment, and to generate one populaton based on data before and after the treatment of each sample (Feldman and Davdson, 1966; Beyer et al., 1967; Kawakam and Sato, 1967; Pfaff and Pfaffman, 1969; Feldman and Dafny, 1970). Ths assumes that the recordng preparaton s otherwse stable durng the latency of the hormone effect. The dstnctons between these two approaches are clearest n the analyss of sngle- or multunt actvty. Jn the two-populaton approach one must assume that there exsts consderable dfferentaton of functon among neurons at partcular stereotaxc coordnates, so large samples must be taken to assume homogenety. In the one-populaton, before and after approach, one s rarely certan that the same neuron or neurons are beng recorded from for perods longer than several hours, especally f the neuronal actvty s changng slowly. It s, therefore, desrable to record the change n actvty of neurons to rapdly-actng hormones. In any case, the problem of determnng "specfcty" s extremely complcated, for t can refer to (1) whether the actvty of one regon but not another s affected (excted or nhbted) by a hormone; (2) whether one hormone but not another affects ths regon but not another regon; (3) whether one stmulus modalty but not another affects one regon but not another and whether one hormone but not another affects ths response. Short-Term Hormonal Effects Relatvely short-term effects on neural actvty have been demonstrated for essentally all the hormones {see Beyer and Sawyer, 1969, for revew). The end result of certan neuroendocrne processes can be changed qute rapdly by hormones, and therefore these are potentally of value n neurophysologcal analyss of hormonal effects. For example, progesterone facltates matng responses wthn mnutes of ntravenous njecton n estrogen-prmed rats (Lsk, 1960) or njecton nto the lateral ventrcle (Kent and Lberman, 1949). Wthn 4-6 hours, progesterone admnstered systemcally sgnfcantly ncreases the proporton of rats whch ovulate n response to electrcal stmulaton of the basal hypothalamus (Docke and Dorner, 1969). Sawyer and Everett (1959) showed that the proporton

10 750 BARRY R. KOMISARUK of estrous or estrogen-prmed (but not anestrous) rabbts whch ovulate n response to vagnal stmulaton was markedly ncreased f progesterone (2 ng) was njected subcutaneously 1-4 hours before stmulaton. Releasng factors stmulate release of ptutary hormones wthn mnutes after ntravenous njecton; however, these effects are probably exerted drectly on the ptutary cells (McCann et al., 1968). Estrogen can elct sexual receptvty n rats wthn as lttle as 24 hours after ntroducton nto the hypothalamus (Chambers and Howe, 1968) or 17 hours after ntravenous njecton of an aqueous suspenson (Green et al., 1970). Vowles and.harwood (1966) reported an ncrease n the proporton of ntact female rng doves showng defensve threat behavor (wng slappng, puffng up, and feather erecton toward an ntruder) wthn 30 mnutes of an aqueous ntramuscular njecton of estradol monobenzoate, testosterone proponate, or prolactn, but not jsrogesterone or salne. The observatons that speces-characterstc behavoral and hormonal effects are produced by these treatments ncreases the lkelhood that they may approach naturally-occurrng events. In the absence of evdence of behavoral, hormonal, or other physologcal changes nduced by hormonal admnstraton, rapd alteratons n bran actvty followng admnstraton are of consderable nterest, but ther functonal sgnfcance s obscure. Tme-Related Ncuroendocrne Events Crtchlow (1956) presents records showng bursts of actvty n the lateral hypothalamc EEG of the rat durng the "crtcal perod" for release of ovulatng hormone. Consstent wth ths, Kawakam et al. (1970) report ncreased multunt actvty n the basal hypothalamus and preoptc regon durng the crtcal perod for ovulaton (and durng the afternoon each day of the estrous cycle). Terasawa and Sawyer (1969) found that n rats n whch electrochemcal stmulaton of the preoptc area nduced ovulaton (despte pentobarbtal blockade of spontaneous ovulaton), multunt actvty n the medan emnence was actvated wth a mean latency of 9 mnutes and a duraton of 30 mnutes. In cases n whch ovulaton faled to occur n response to the stmulaton, the multunt actvty n the medan emnence showed no ncrease. Progesterone admnstraton at 11:00 n natural proestrus actvates release of ptutary ovulatng hormones; multunt actvty n the medan emnence at ths tme of day ncreased ntally n response to progesterone whereas at 14:30 t decreased ntally. The ntal rse wth early progesterone was blocked by transectons between the preoptc area and the medan emnence (Terasawa and Sawyer, 1970). These fndngs, usng combned technques, effectvely relate neural events n tme and place to objectve physologcal endponts. Brooks et al. (1966) have shown n cats that artfcal sucklng stmulaton or uterne dstenson leads wthn seconds to an ncrease n ntramammary pressure, ndcatve of oxytocn acton; ths s preceded by an ncrease n frng rate of neurons n the paraventrcular nucleus, whose neurons secrete oxytocn. Faure et al. (1967) have recorded ncreased neuronal dscharge rate n the premamllary regon of the basal hypothalamus (and not n the dorso-ventromedal hypothalamc area) as early as one mnute after electromechancal stmulaton of the vagna n rabbts, suggestng that ths regon may be nvolved n the reflex release of ovulatng hormone. CONCLUDING REMARKS Neuronal recordng can provde nformaton on (1) the stes, tme, and specfcty of acton of hormones on the nervous system; (2) the source, magntude, and modfablty of neural nput to partcular neurons from dstant regons; (3) the bran output mechansms to whch partcular neurons are related; (4) the relatonshp between hormonal acton and neurotransmtter acton; (5) the relatonshp of neuronal frng to neurosecreton; (6) stes of acton of drugs on neuroendocrne and J \ I

11 NEUROENDOCRINE MECHANISMS 751 other bran mechansms; (7) the dfferentaton or "dvson of labor" of varous components of functonal bran systems. The power of neurophysologcal analyss of neuroendocrne processes s ncreased substantally f the relevant functonal anatomy and physologcal output are assessed jontly. REFERENCES Alcaraz, M., C. Guzman-Flores, M. Salas, and C. Beyer Effect of estrogen on the responsvty of hypothalamc and mesencephalc neurons n the female cat. Bran Res. 15: Ara, Y., M. Hro, J. Mtra, and R. A. Gorsk Influence of ntravenous progesterone admnstraton on the cortcal electroencephalogram of the female rat. Neuroendocrnology 2: Aron, C, G. Asch, and J. Roos Trggerng of ovulaton by cotus n the rat. Int. Rev. Cytol Barraclough, C. A Hypothalamc actvaton assocated wth stmulaton of the vagnal cervx of proestrous rats. Anat. Rec. 136:159. Barraclough, C. A., and B. A. Cross Unt actvty n the hypothalamus of the cyclc female rat: Effect of gental stmul and progesterone. J. Endocrnol. 26: Beyer, C, V. D. Ramrez, D. I. Whtmoyer, and C. H. Sawyer Effects of hormones on the electrcal actvty of the bran n the rat and rabbt. Exp. Neurol. 18: Beyer, C, and C. H. Sawyer Hypothalamc unt actvty related to control of the ptutary gland, p In W. F. Ganong, and L. Martn [ed.], Fronters n neuroendocrnology. Oxford Unv. Press, New York. Brooks, C. McC, T. Ishkawa, K. Kozum, and H-H. Lu Actvty of neurones n the paraventrcular nucleus of the hypothalamus and ts control. J. Physol. (London) 182: Chambers, W. F., S. L. Freedman, and C H. Sawyer The effect of adrenal sterods on evoked retcular responses. Exp. Neurol. 8: Chambers, W. F., and G Howe A study of estrogen-senstve hypothalamc centers usng a technque for rapd applcaton and removal of estradol. Proc. Soc. Exp. Bol. Med. 128: Crtchlow, B. V Electrcal correlates of neuroendocrne actvty. Psychat. Res. Rep. 6: Cross, B. A Electrcal recordng technques n the study of hypothalamc control of gonadotrophn secreton, p In Proc. 2nd Int. Congr. Endocrnol. Amsterdam: Excerpta Medca Int. Congr. Ser. No. 83. Cross, B. A., and R. G. Dyer Does oxytocn nfluence the actvty of hypothalamc neurones? J. Physol. (London) 203:70-71P. Cross, B. A., and I. A. Slver Effect of luteal hormone on the behavor of hypothalamc neurones n pseudopregnant rats. J. Endocrnol. 31: Cross, B. A., and I. A. Slver Electrophysologcal studes on the hypothalamus. Brt. Med. Bull. 22: Dafny, N., and S. Feldman Sngle cell actvty n the hypothalamus n ntact and adrenalectomzed rats. Physol Behav. 5: Dempsey, E. W., and H. F. Searles Envronmental modfcaton of certan endocrne phenomena. Endocrnology 32: Docke, F., and G. Dorner A possble mechansm by whch progesterone facltates ovulaton n the rat. Neuroendocrnology 4: Endrocz, E Neural and hormonal regulaton of the patterns of sexual behavor, p In K. Lssak [ed.], Symposum on reproducton. Congr. Hung. Soc. Endocrnol. Metab. Akadema Kado. Budapest. Endrocz, E Effect of sex hormones on the electrcal actvty of bran stem and dencephalon n castrated female rats. Acta Physol. Acad. Sc. Hung. 35: Everett, J. W Preoptc stmulatve lesons and ovulaton n the rat: "Thresholds" and LH-release tme n late destrus and proestrus, p In E. Bajusz and G. Jasmn [ed.], Major problems n neuroendocrnology. S. Karger, New York. Everett, J. W Provoked ovulaton or longdelayed pseudopregnancy from cotal stmul n barbturate-blocked rats. Endocrnology 80: Faure, J. M., J. D. Vncent, C. L. Bensch, B. Favarel- Garrgues, and B. Duty Actvtds el^mentares dans l'hypothalamus an cours de 1'ovulaton chez la lapne chronque. J. Physol. Exp. Neurol. 27: Feldman, S., and N. Dafny Effects of corlsol on unt actvty n the hypothalamus of the rat. Exp. Neurol. 27: Feldman, S., and J. M. Davdson Effect of hydrocortsone on electrcal actvty, arousal thresholds and evoked potentals n the brans of chroncally mplanted rabbts. J. Neurol. Sc. 3: Feldman, S., and Y. Same Effect of cortsol on sngle cell actvty n hypothalamc slands. Bran Res. 23: Fox. S. S., and J. H. O'Bren Duplcaton of evoked potental waveform by curve of probablty of frng of a sngle cell. Scence 147: French, J. D., M. Verzeano, and H. \V. Magoun A neural bass of the anesthetc state. AMA Arch. Neurol. Psychat. 69: Fromm, G. H., and \V. H. Bond The relatonshp between neuron actvty and cortcal steady potentals. Electroencephalogr. Cln. Neuro-

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