Prolactin and luteinizing hormone release after diencephalic lesions and stimulation

Brain ResearchBulletin. Vol. 6, pp. 243--249, 1981. Printed in the U.S.A.

Prolactin and Luteinizing Hormone Release After Diencephalic Lesions and Stimulation I J. A. C O L O M B O t A N D C. P. P H E L P S D e p a r t m e n t o f A n a t o m y , University o f South Florida, Tampa, FL R e c e i v e d 18 O c t o b e r 1980 COLOMBO, J. A. AND C. P. PHELPS. Prolactin and luteinizing hormone release after dieacephalic lesions and stimulation. BRAIN RES. BULL. 6(3) 243-249, 1981.--Adult male rats received sham procedures or retrochiasmatic knife cuts of varying sizes. Ten to twelve days (short term survival) or 90 days (long term survival) later animals were placed under pentobarbital anesthesia and pulsed, monophasic (50 Hz, 200 p.A, 0.5 msec width, 30 sec on/off) stimuli were delivered during 30 min through bipolar, concentric electrodes bilaterally placed in the medial preoptic-suprachiasmatic nucleus (MPOA-Sch). Blood samples were taken before and at 30 min intervals after stimulation. Plasma prolactin and LH were measured in the same sample. Penetration of the knife guide tube induced a lesion of cortical and subcortical midline structures (sham procedures) placed at or around AP plane 5.0 of the Atlas of Albr-Fessard et al. This resulted in a short term impairment of LH release induced by MPOA-Sch stimulation, an effect that could not be distinguished from the one observed after frontal cuts made with a knife having a 1.5 mm radius (1.5 FC). Frontolateral retrochiasmatic cuts (LFC) blocked the effects of stimulation on LH release. After 90 days, LH response to MPOA-Sch stimulation improved in sham FC and 1.5 FC groups. Sham procedures did not reduce prolactin response after short term survival. 1.5 FC significantly decreased and LFC blocked prolactin release response. Ninety days after FC there was no improvement of prolactin release response to MPOA-Sch stimulation. In conclusion, long term survival after sham and 1.5 FC surgical procedures allowed for a partial recovery of the LH release response, but not that of prolactin, after MPOA-Sch stimulation. Prolactin

LH

Hypothalamus

Preoptic area

P U L S E D electrical stimuli applied through bipolar electrodes placed within the ventral and medial aspects of the medial preoptic-suprachiasmatic region (MPOA-Sch) in male rats is known to be an efficient stimulus for the induction of LH and prolactin release from the anterior pituitary [5]. Other parameters being constant, the comparative efficiency of such stimulation to release LH or prolactin was shown to be partially a function of the applied pulse rates within a certain range [6]. Also, for a given set of stimulation parameters the shape of the prolactin release response curve, unlike that of LH, depended upon the electrode location within the medial preoptic area-hypothalamic continuum. The suggestion was made that under the conditions tested, a lack of differences in stimulated-induced release of LH within this region was indicative of the existence of sets of control elements with similar functional characteristics, as opposed to the case of prolactin [6]. Transection of axonal processes results in degeneration of nerve terminals. This procedure provides a means of suppressing, at least temporarily, active conduction in selected fibers passing through a volume of stimulated tissue as well as of locally generated signals. If enough time is allowed, this impairment may be partially overriden by neural regeneration, collateral sprouting, and/or the establishment of new synaptic connections, which may be associated with development of denervation hypersensitivity phenomena. The

Functional recovery technique of surgical deafferentation of discrete brain areas has provided meaningful information in neuroendocrine studies [9, 1 l, 12, 20] designed to establish those functions that remain unimpaired or those being suppressed. By combining the effects of electrical stimulation and deafferentation techniques on the release of LH, prolactin and TSH, the present studies were intended to gain further information on anatomical and functional grounds for the differential control of each hormone by the POA-hypothalamic complex. Results regarding TSH are reported elsewhere [24]. These data have been reported previously in abstract form [22]. METHOD For the most part, the procedures have been fully described in another paper to which the reader is referred [24]. For practical purposes, a brief description follows. Adult Holtzman male rats were kept under a 14 hr lights on-10 hr lights off regime. Several control and experimental groups were studied (average of 7 animals per group). A retractable Hal~isz-type knife with a 1.3 or 1.5 mm radius was used to produce retrochiasmatic frontal cuts (1.3 or 1.5 FC) using the Alb6-Fessard, Stutinksy and Libouben atlas [2]. The 1.5 mm radius knife was also used to produce an extended frontolateral cut (LFC). A third experimental group consisted of low-

tPartial support for this work was provided by Grants PCM-7096284 (NSF), Biomedical Research Support Grant NIH 5-SO7 RR-5749 and USPHS HD11345. -"Send reprint requests to J. A. Colombo, Department of Anatomy, University of South Florida, College of Medicine, Box 6, 12901 N. 30th Street, Tampa, FL 33612.

C o p y r i g h t © 1981 A N K H O I n t e r n a t i o n a l Inc.--0361-9230/81/030243-07501.20/0

2~

COLOMBO A N D P H E L P S

S H O R T T E R M = io-r2 DAYS LONG TERM =9ODAYS

A

B

7.0 60. 5.0. 13 FC 4.0

2'.o ~o &

LONG TERM

1.SFC I

1I"sLFc

1.SFCJ SHORT TERM

FIG. 1. (A) Diagram showing the relative position of one electrode to the knife cut on a parasagittal plane [2]. (B) Reconstruction of each knife cut according to size, location and excursion, projected on a hypothetical plane (adapted) from AIb6-Fessard, Stutinsky and Libouban [2].

ering the assembly and producing a midline lesion by only extruding the knife in the median sagittal plane (MC). Three control groups were produced: intact (I) and sham procedures for either the FC (Sham FC) or the LFC (Sham LFC) type surgery. The Sham FC procedures consisted in lowering (5.5 mm) the guide tube of the knife through the midline. Sham LFC animals were subjected to similar procedures, plus a 2.8 mm caudorostral displacement of the carder. Ten to twelve days (short term survival) or 90 days (long term survival) after surgery, animals were anesthetized with pentobarbital (35 mg/kg IP), tracheotomized and a femoral artery cannulated for sample withdrawal (0.9 ml). Blood cells were returned to the animals after being resuspended in saline. Approximately nine hours elapsed between the injection of pentobarbital and the time at which the first sample was withdrawn. Stimulation was applied through concentric stainless steel electrodes placed in the MPOA-Sch region following the de Groot atlas coordinates [8]. Stimulation consisted of monophasic, tip cathode, rectangular 0.5 msec width pulses, 200/zA, 50 Hz, 30 sec on/off during a 30 min period. Current delivered through each electrode was monitored in an oscilloscope (Tektronix T912). At the end of the experiment, 10--15 p.A, 15 sec anodal direct current was passed through the electrode tips for marking purposes. Perfusion with K ferrocyanide and 10% Formalin allowed for the determination of electrode location in 40 p,m frozen sections,

that were counterstained with cresyl violet. Brains were histometrically analyzed to establish the relative placements of electrodes and knife cuts. Plasma samples were stored at -70°C until assayed using radioimmunoassay procedures (NIAMDD). Although most samples were measured in the same assay, when this was not the case, a common reference standard was used to normalize the data. Values were expressed in ng/ml of Rat LHRP-1 and Rat Prolactin RP-I, Data was analyzed using the analysis of variance, SAS means procedure, and paired t tests (SAS-GLM program [251).

RESULTS As shown previously in intact male rats under pentobarbital anesthesia, the average concentration of LH and prolactin in blood plasma remains essentially at a rather constant level in the absence of brain stimulation [5]. The brain midline region lesioned by the sham procedures partially included the cerebral cortex, corpus callosum, fimbria, stria medullaris, habenula, and part of the thalamus [24]. The extent of knife cuts (1.3, 1.5 FC, LFC) and the relative location of the stimulating electrode with respect to the knife cuts are presented in Fig. 1. Further details regarding the histological histometric analysis of electrode pair placements and their distance to the knife cuts are given in the accompanying paper [24].

INDUCED PROLACTIN AND LH RELEASE

245

B

A

(~ SHORT TERM SURVIVAL INTACT o-o 1.5 FC H SHAM FC

400 =

400-

I

300. A

O'SHORT TERM ~ A L ~-A INTACT o-.o LFC SHAM LFC

300-

200.

n200-

4n ,< 100100-

,..,,,.. !

& .......

~

~o

TIME(m/n)

TIME(rnin)

(~

go

OdLONG TERM SUkqV]VAL k-a, INTACT H SHAM FC

D 4~0-

4OO"

300-

E

5 1E

200-

I

" 04 L.ONGTERM 8UlTV~A~L D.e MC: 1.3 FC

O.(] 1.5 FC

11111-

100"

1

~f

I o ...... TiME (n~n)

20 TIME (mini

FIG. 2. LH release after MPOA-Sch stimulation in various experimental groups. Arrows indicate extent of stimulation. Mean values±SE. A and B = 12-14 days survival after surgery, C and D=90 days survival. (A, B) Note similar effects in sham FC, 1.5 FC and sham LFC. Also, the complete blockade of POA-Sch stimulation of LH release in LFC rats. (A, D) Observe the relatively larger increase in 1,5 FC after 90 days from surgery as compared to the short term survival.

246

COLOMBO AND PHELPS

( ~ S H O R T TERM SURVIVAL

A

(~ISHORT TERM SURVIVAL ~,~ LFC O ' a SHAM LFC • -~ iNTACT

B

--, ~rA~

o-~ 1.5 FC H S H A M FC 400"

J

E

400-

300-

300-

200-

200-

m <[ .J G. 100-

1004

I

6

TIME (mln)

~o

STIMAJI-&TION

~

,~.

~o

TiME ( min )

¢~ ~ C

H

TERN SURVIVAL iNTACT SHAM FC

O* LONG TERM SURVWAL HMC 1.3 FC D-o t $ FC

D 400-

E

300-

i

'

_z

!

! 200-

100-

100"

do TIME (rain)

~

........

3'o

do

&

TIME (rain)

FIG. 3. Prolactin release after MPOA-Sch stimulation in various experimental groups---same as in Fig. 2. (A, B) Note that 1.5 FC and LFC greatly reduced the release of prolactin after stimulation, but sham procedures did not (as opposed to LH). (A, D) Observe no improvement with time in the ability of MPOA-Sch stimulation to release prolactin after 1.5 FC.

INDUCED PROLACTIN AND LH R E L E A S E 0~0

300'

ALH

.' TS L

I

1

,,,,"

200' Z 0 e3

247

100-

ent in this group 30 min after stimulation (/)<0.05), while in LFC animals, the prolactin response to stimulation was completely blocked (Fig. 3B). After long term surgery in MC, 1.3 and !.5 FC groups there was a slight increase in basal prolactin levels (p=0.05; <0.05; <0.05, respectively) without a corresponding increase in poststimulation levels over those present in intact animals. These increased basal levels resulted in a flattened response when compared w'ith the intact group (Figs. 3C, D). However, in spite of this attenuated response, significant increases from 0 time levels occurred in 1.3 (,o<0.05) and 1.5 (p<0.05) FC groups. The response in the latter group did not differ from the one observed in 1.5 FC short term animals. Post-stimulation increase in i.3 and 1.5 FC groups was similar to that present in MC animals, with FC groups showing a comparatively reduced response when compared to that of the intact group. DISCUSSION

IN4ACT

MC

1.3 I=C

1..5I=C

SURGERY

FIG. 4. Comparative effects of MPOA-Sch stimulation on LH, prolactin and TSH (data taken from [24]) blood titers at 30 min after onset of stimulation as a function of brain damage (90 days postoperative period). Values are expressed as the differential between 30 and 0 rain for each hormone. MC=midline cut; 1.3 FC=frontal cut with 1.3 mm blade radius; 1.5 FC=frontal cut with 1.5 mm blade radius.

Plasma LH levels were significantly increased (p =0.0035) 30 min after stimulation of the MPOA-Sch region in intact male rats. Levels decreased thereafter, reaching control values by 90 min (Fig. 2A). Short term FC and LFC procedures resulted in a lowering of the maximum levels attained by 30 min (Fig. 2A, B). In 1.5 FC animals, there was a reduced rsponse at 30 min (p<0.01) when compared to I rats, but levels were still significantly higher than those at 0 time (/9<0.01). A similar change was also observed in the short term FC sham group. On the contrary, in LFC rats the release response to MPOA-Sch stimulation was completely blocked, whereas sham LFC had no significant effect on the 30 min LH response, although maximum levels were somewhat reduced (Fig. 2B). In long term MC, 1.3 FC and 1.5 FC lesioned animals, stimulation resulted in a LH response of amplitude similar to that present in intact rats and mean plasma LH concentrations of cut rats remained comparatively higher than in the intact group at 60 and 90 min. The effects of sham surgical procedures did not result in significant changes with time between short and long term operated animals (Figs. 2A, C). An additional interesting finding was that the response to stimulation in the 1.5 FC groups improved with time (p<0.01 for differences between short term 30 rain & and long term 30 rain A) (compare Figs. 2A, D). As shown in Fig. 3A, prolactin response after short term sham procedures did not differ significantly from that present in intact rats. Prolactin levels were increased by 30 min (p<0.01) and decreased toward 0 time levels by 90 min (p<0.05). On the contrary, short term 1.5 FC and LFC procedures resulted in a significant decrease (p<0.05 vs. intact) in the ability of POA-Sch stimulation to increase plasma prolactin levels (Fig. 3A, B). In spite of this decrement in short term 1.5 FC animals, a slight increase was still appar-

The present results confirm those obtained in previous studies in which electrical, pulsed stimuli applied within the MPOA-Sch region induced a significant increase in plasma LH and prolactin concentration in intact male rats [5,6]. Although stimulation at 10 Hz resulted in maximum responses for prolactin, when other parameters were held constant, this was not the case for LH, which was maximally released with a pulse frequency of 50 Hz [5,6]. Also in those studies, maximum average values were obtained at 30 min for intact animals and, at variance with growth hormone [15], no evidence of a 'rebound' phenomenon was apparent that could be interpreted as post-inhibitory release. In order to more adequately discuss the present results, the problem of the possible extent of the applied electrical field should be considered first. The average placement of the stimulating electrode tips (cathode) with respect to the edge of the knife cut was 0.8 mm. Since the electrodes were inserted at an angle with respect to the knife (Fig. I), the exposed barrel (anode) was at an even greater average distance from the cuts. Consequently, the main axis of the volume of tissue directly affected by the stimulation was obliquely oriented in a caudal-rostral direction from the electrode tips. An estimation of the actual current spread with bipolar electrodes [7] would suggest that there was a low probability that elements caudal to the knife cut would have been directly stimulated at full intensity. Moreover, our observation that in short term FC rats the hormonal release response was greatly reduced, and that in LFC animals the release response for both hormones was almost blocked could be taken as an indication that direct stimulation of neural elements caudal to the cut were not activated in a functionally significant manner. This assumption forms the basis of this discussion and of that in the accompanying paper [24]. Although sham FC and 1.5 FC procedures similarly reduced LH release response to MPOA-Sch stimulation, this reduction was probably related to the effects of a common. non-specific trauma that masked the functional effects of inherent structural changes induced by each of those procedures. According to several authors, LHRH perikarya are located in the MPOA-Sch region [1, 3, 32] and their processes end in terminals in the arcuate nucleus-median eminence region. In addition, the presence of LHRH cells located in the tuberoinfundibular region has also been proposed [13,27]. Based on the similar response profile of LH concentration in plasma following electrical stimulation of the MPOA-Sch or the VMH-arcuate nucleus regions with

248

COLOMBO AND PHELPS

two different frequencies, the suggestion was made that a similar set of elements was stimulated [6], probably the L H R H pathway. We also observed, however, an increase in plasma L H after stimulation in the 1.5 FC rats, which was blocked in LF C animals. This residual capacity to respond to stimulation after a frontal cut was made in the retrochiasmatic area may indicate the involvement of a more anterolateral input to elements in the mediobasal hypothalamus controlling the release of LH, or of fibers coursing dorsal to the cut [26]. Since after 10-14 postoperative days one would expect complete degeneration of the axons, including those containing LHR H, the residual effect could be related to the proposed second population of L H R H perikarya in the MBH. If so, it would be necessary to accept the corollary that this second population of L H R H neurones had probably been activated transynaptically. The existence of an anterolateral, or lateral input (mostly MFB) to the medial hypothalamus has been addressed on morphological and electrophysiological grounds [19, 20, 28, 29] and its possible contribution to the control of LH release has also been discussed [4, 16, 21]. Partial recovery Of LH release was observed in FC rats after an extended postoperative period of 90 days. Although partial regeneration of L H R H axons may have occurred, the effects of surgery and postoperative intervals on MBH LHRH-containing neurons are unknown. The possibility of new synaptic connections or altered functional (synthesis, storage and release) arrangements for L H R H neurons should also be considered. Short term survival after sham surgical procedures did not result in a reduction of prolactin release response to MPOA-Sch stimulation. Frontal cuts produced a significant reduction in prolactin release response with a rather small residual capacity to respond to stimulation. As was the case

for LH release, LFC animals showed a complete blockade of prolactin release response. Both inhibitory and releasing factors have been proposed to regulate the release of pituitary profftctin (for reviews see [14, 17, 31]). While it remains to be established whether the induction of prolactin release after MPOA-Sch stimulation is causally related to changes in the activity of prolactin-inhibitory and/or releasing elements, it seems likely that effects on a presumed dopamine mediation after retrochiasmatic knife cuts are not related to decreased dopamine availability [23,32], but possibly to synaptically mediated effects on dopamine neurons. Finally, it should be stressed that the different responses obtained for LH, prolactin and TSH [22,24] after stimulation of the MPOASch region were related to the extent of "midline cut damage" (see Fig. 4 for comparisons). It seems clear that although the TS H response was facilitated by increasing brain damage, the prolactin response was progressively reduced and the LH response remained unchanged in those same groups. The combination of deafferentation and stimulation procedures offers another approach to the study of neural mechanisms underlying the segregation of functions within a complex region such as the MPOA-hypothalamic continuum. Further studies are being carried out exploring some questions arising from the present experimental results.

ACKNOWLEDGEMENTS The authors would like to express their appreciation to Donna Anderson and Paul Luth for technical assistance and for the illustrations, and Betsey Stiepock and Rozena Stanage for secretarial work. The authors are greatful to Jim Moore, Ph.D. (Computer Research Center, USF) for assistance in statistical analysis.

REFERENCES 1. Ajika, K. Frontiers in Nenroendocrinology, Volume 6. New York: Raven Press, 1980, pp. 1-32. 2. Albr-Fessard, D., F. Stutinsky and S. Libouban. Atlas stdr~otaxique du dienc~phale du rat blanc. Paris: C.N.R.S., 1966. 3. Barry, J., M. P. Dubois and P. Poulain. LRF producing cells of the mammalian hypothalamus. A fluorescent antibody study. Z. Zellforsch. 146: 351-366, 1973. 4. Carrer, H. F. and S. Taleisnik. Neural pathways associated with the mesencephalic inhibitory influence on gonadotropin secretion. Brain Res. 38: 299-313, 1972. 5. Colombo, J. A. Prolactin and LH release after stimulation of the preoptic-suprachiasmatic and arcuate-ventromedial n. regions in male rats under pentobarbital anesthesia. Neuroendocrinology 26: 150-162, 1978. 6. Colombo, J. A. Preoptic-hypothalamic control of LH and prolactin release in male rats under pentobarbital anesthesia: differential responses to electrical stimulation. Brain Res. 144: 313-323, 1978. 7. Crawley, J. N., J. W. Maas and R. H. Roth. Evidence against specificity of electrical stimulation of the nucleus locus coeruleus in activating the sympathetic nervous system in the rat. Brain Res. 183: 301-311, 1980. 8. de Groot, J. The rat forebrain in stereotaxic coordinates. Trans. ry. Neth. Acad. Sci. 52: 1-40, 1967. 9. Hal~isz, B. and L. Pupp. Hormone secretion of the anterior pituitary gland after physical interruption of all nervous pathways to the hypophysiotrophic area. Endocrinology 77: 553562, 1965. 10. -Hal~isz, B. and R. A. Gorski. Gonadotropic hormone secretion in female rats after partial or total interruption of neural afterents to the medial basal hypothalamus. Endocrinology 80: 608622, 1967.

11. Halfisz, B., W. H. Florsheim, N. L. Corcorran and R. A. Gorski. Thyrotrophic hormone secretion in rats after partial or total interruption of neural afferents to the medial basal hypothalamus. Endocrinology 80: 1075-1082, 1967. 12. Kimura, F. and M. Kawakami. Reanalysis of the preoptic afferents and efferents involved in the surge of LH, FSH and prolactin release in the proestrous rat. Neuroendocrinology 27: 74--85, 1978. 13. Krey, L. C. and A. J. Silverman. The luteinizing hormonereleasing hormone (LH-RH) neuronal networks of the guinea pig brain. II. The regulation of go~aadotropin secretion and the origin of terminals in the median eminence. Brain Res. 157: 247-255, 1978. 14. MacLeod, R. M. Frontiers in Neuroendocrinology, Volume 4. New York: Raven Press, 1976, pp. 16%194. 15. Martin, J. B. Inhibitory effect of somatostatin (SRIF) on the reelase growth hormone (GH) induced in the rat by electrical stimulation. Endocrinology 94: 497-503, 1974. 16. Martinovic, T. V. and S. M. McCann. Effect of lesions in the ventral noradrenergic tract produced by microinjection of 6-hydroxy dopamine on gonadotrophic release in the rat. Endocrinology 100: 1206-1213, 1977. 17. Neill, J. D. Handbook of Physiology. Section 7, Endocrinology, Volume 4. Washington, D.C.: Am. Physiol. Soc., 1979, pp. 469-488. 18. Neill, J. D. Frontiers in Neuroendocrinology, Volume 6. New York: Raven Press, 1980, pp. 12%155. 19. Perkins, M. N. and S. A. Whitehead. Responses and pharmacological properties of preoptic/anterior hypothalamic neurones following medial forebrain bundle stimulation. J. Physiol. 279: 347-360, 1978.

INDUCED PROLACTIN AND LH RELEASE

20. Perkins, M. N. and S. A. Whitehead. Neural connexions between the medial forebrain bundle, the preoptic area and the basal hypothalamus in the rat: an electrophysiological study. J. Physiol. 291: 443--456, 1979. 21. Phelps, C. P. and C. H. Sawyer. Electrochemically stimulated release of luteinizing hormone and ovulation after surgical interruption of lateral hypothalamic connections in the rat. Brain Res. 131: 335--344, 1977. 22. Phelps, C. P. and J. A. Colombo. Combined effects of frontal hypothalamic cuts and preoptic (POA) stimulation on anterior pituitary hormone release. Proc. 9th A. Meet. Soc. Neurosci, 232, 1979. 23. Phelps, C. P., S. Saporta and D. M. Nance. Electrochemically stimulated (ECS) luteinizing hormone (LH) release and nuclear catecholamine (CH) content after hypothalamic knife cuts. Proc. IOth A. Meet. Soc. Neurosci., Abst. 254.1. 24. Phelps, C. P. and J. A. Colombo. Facilitated thyrotropin release after retrochiasmatic hypothalamic knife cuts. Brain Res. Bull. 6: 235--242, 1981. 25. SAS Users Guide, 1979 Edition. Raleigh: SAS Institute, Inc., 1979.

249 26. Setal6, G., S. Vigh, A. V. Schally, A. Arimura and B. Flerk6. Immunohistological study of the origin of LH-RH-containing nerve fibers of the rat hypothalamus. Bra#~ Res: I03: 597-602, 1976. 27. Silverman, A. J. and L. C. Krey. The luteinizing hormonereleasing hormone (LHRH) neuronal networks of the guinea pig brain. I. lntra- and extrahypothalamic projections. Brain Res. 157: 233-246, 1978. 28. Swanson, L. W. and W. M. Cowan. The efferent connections of the suprachiachiasmatic nucleus of the hypothalamus. J. comp. Neurol. 160: 1-12, 1975. 29. Swanson, L. W. An autoradiographic study of the efferent connections of the preoptic region in the rat. J. comp. Neurol. 167: 227-256, 1976. 30. Tejasen, E. and J. W. Everett. Surgical analysis of the preopticotuberal pathway controlling ovulatory release of gonadotropins in the rat. Endocrinology 81: 3187-3196, 1967. 31. Tindal, J. S. The Endocrine Hypothalamus. New York: Academic Press, 1978, pp. 333-360. 32. Weiner, R. I., J. E. Shryne, R. A. Gorski and C. H. Sawyer. Changes in the catecholamine content of the rat hypothalamus following deafferentation. Endocrinology 90: 867-873, 1972. 33. Zimmerman, E. Frontiers in Neuroendocrinology. New York: Raven Press, 1976, pp. 25-93.