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Preoptic-hypothalamic control of LH and prolactin release in male rats under pentobarbital anesthesia: Differential responses to electrical stimulation
Brain Research, 144 (1978) 313-323 © Elsevier/North-HollandBiomedicalPress
PREOPTIC-HYPOTHALAMIC
CONTROL
313
OF
LH
AND
PROLACTIN
RELEASE IN MALE RATS UNDER PENTOBARBITAL ANESTHESIA: DIFFERENTIAL RESPONSES TO ELECTRICAL STIMULATION
JORGE A. COLOMBO* Instituto de Investigaciones Cerebrales, Facultad de Ciencias Af~dicas, Universidad Nacional de Cuyo, Mendoza (Argentina}
(Accepted August 3rd, 1977)
SUMMARY In acute experiments under pentobarbital anesthesia, the brains of adult male rats were stimulated bilaterally with trains of monophasic pulses at different frequencies and current intensities through bipolar, concentric, stainless steel electrodes. Stimulation of the POA-SQ region at 3, l0 (200/~A) or 200 Hz (100/~A) increased plasma prolactin levels within 30 min of the onset of stimulation. Stimulus parameters fully effective when applied to the POA-SQ or ARC-MED. EM. regions were ineffective when applied to the ARC-VMH region. Maximal effects were obtained with 10 Hz (200/zA) stimuli applied to the POA-SQ region. LH levels were increased after stimulation of any of the three regions - - POASQ, ARC-VMH or ARC-MED. EM. Conversely to the prolactin response, LH levels did not rise after 3 Hz stimulation of the POA-SQ region. In male rats under pentobarbital anesthesia, LH levels seemed to be affected maximally with a stimulus frequency rate of about 50 Hz applied to the POA-SQ region. The results suggest a potential physiological role of frequency discrimination in the functional segregation of LH and prolactin release controls.
INTRODUCTION The preoptic-hypothalamic complex contains neural circuits controlling pituitary prolactin and luteinizing hormone (LH) release. Stimulation studies have been concerned primarily with the localization of circuits controlling the release of indivi* Member of the ResearchCareer of the ConsejoNacional de InvestigacionesCientificasy T6cnicas (Argentina). Present address: Department of Anatomy, College of Medicine, Medical Center,
Box 6, 12901 North 30th Street, Tampa, Fla. 33612, U.S.A.
314 dual hormones. The dense packing of neural elements suggests that several factors might be operating to assure a given set of outputs for a corresponding set of inputs. Among them, frequency response is a possible way to discriminate functions within a given neuronal population 15A9. A wide range of spontaneous firing frequency rates has been described in the rat for units located within the preoptic-hypothalamic complex5,1°,11,22,23,2s, yet they rarely exceed 10 spikes/sec. At least in the final common path of the neuroendocrine effector system information transfer appears to bear a direct relationship to changes in spike density17. The end result would thus depend essentially on changes in the number of potentials arriving at the final (effector) element during a given period. The existence of 'silent' and extremely slow firing units implies that a functionally significant increase in firing rates could be attained with what has traditionally been considered low frequency stimulation. Data presented in this paper yield evidence that elements controlling release of LH and prolactin within the preoptic-suprachiasmatic nucleus (POA-SQ), arcuate nucleus-ventromedial nucleus (ARC-VMH) and arcuate nucleus-median eminence (ARC-MED. EM.) regions do not share the same responses to a given set of applied electrical stimuli, and that they are sensitive to low frequency stimulation. Furthermore, data are consistent with the hypothesis that frequency discrimination is a possible mechanism for the separate control of release of the two hormones. MATERIAL AND METHODS Adult Holtzman male rats (250-330 g) maintained under controlled illumination (lights on 06.00-20.00 h) and fed with standard laboratory diet and water ad libitum were used. Animals were anesthetized with pentobarbital (35 mg/kg, i.p.). A tracheotomy was performed and a femoral artery cannulated for blood sample withdrawal. The animal was then placed in a stereotaxic instrument where it remained until the completion of the experiment. Animals were maintained warm with the aid of an incandescent bulb. A stainless steel, bipolar, concentric electrode pair was directed to preoptic or hypothalamic locations following the De Groot atlas 9 coordinates. Electrodes were made with 25 g tubing and 150/~m insulated inner wire, with similar characteristics to those used by Terasawa and Sawyer26. Bilateral electrical stimulation consisted of monophasic (outer barrel anode, inner wire cathode) rectangular pulses generated by an $4 Grass stimulator and driven through a variable resistor box placed in series with the preparation. Current intensity was continuously monitored through an oscilloscope and readjusted when needed. Standard parameters were: 0.5 msec width, 30 sec trains every other 30 sec, 100-200/zA current intensity, 3-10200 Hz, for a period of 30 min. Stimulation started 45 min after the electrode placement and not less than one hour after pentobarbital administration. After an initial (0 time) prestimulation sample (0.9 ml) was taken, blood sampling continued every 30 min during a total period of 90 min. Following blood centrifugation and plasma separation, blood cells were resuspended in physiological s~line ~nd injected back
315 after the next sample. At the end of the experiment electrode locations were established by means of the Prussian blue reaction after passing marking current through the electrode tips. Brains were perfused with K ferrocyanide and 10 ~ formalin and 80/~m frozen sections examined in a dissecting microscope. Plasma samples were stored frozen at --20 °C until radioimmunoassay procedures. L H and prolactin were measured in the same plasma sample by the double antibody radioimmunoassay technique, using the kits supplied by Dr. A. F. Parlow (NIAMDD, NIH). Results are expressed in ng/ml of plasma of the N I A M D D RatLH-RP 1 and N I A M D D Rat-Prolactin-RP 1. Analysis of variance 4 was used to compare differences between and within groups. RESULTS Sham-operated animals showed small, non-significant hormonal variations across time, with a tendency to give higher values by the end of the experimental period. Control (0 time) values of each experimental group did not differ significantly from those obtained in unstimulated controls. d t rat
150-
POA-SQ
(acute)
Prl I P - - 4 LH 0 - - - - 0
3Hz,200uA
Prl -
2 o o H z , l o o uA
LH
-
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100-
c Z
0o)
F-
--
(6)
050-
(e)
IL
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I
I
I
0
30
60
90
Time
(rain)
Fig. 1. Bilateral stimulation of the POA-SQ region with 3 Hz (200/~A) or 200 Itz (100 ~A) upon LH and prolactin plasma levels in male rats under pentobarbital anesthesia. Mean 4- S.E. Figures in brackets = number of animals per group. S, period of stimulation.
316
Odrat
150-
lo Hz
(a c u t e )
uA,
O--"O
100
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200 uA,
POA--SO
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100-
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\
/
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/
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o 50-
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0
30
60
90
Time (mi~
Fig. 2. Effects of l0 Hz stimulation on plasma prolactin levelsin male rats under pentobarbital anesthesia. Same asin Fig. 1. Stimulation of the POA-SQ region at 3 Hz (200 FA) affected prolactin but not LH plasma levels, except for the 90 min sample (Fig. 1). Prolactin values increased by 30 min, returned to control levels by 60 min and tended to give high values in the 90 min sample. Overall differences with the sham group were statistically significant (P < 0.01) and also at 30 min (P < 0.01) and 90 min (P < 0.01), and so were within group differences (P < 0.05), with 0 vs. 30 min P < 0.01 and 30 vs. 60 min P < 0.05. Individual increases (defined here as a 100~ increase above control level at 30 or 60 min) were observed in 6 out of 10 animals. On the contrary, L H levels remained practically unchanged (only 2 out of 12 animals produced a significant increase) except fora late increase by the 90 min sample (0 vs. 90 min P < 0.05) (Fig. 1). Most electrode placements were localized at midventral sites of the MPO and AHA, in some cases involving one of the suprachiasmatic nuclei. With l0 Hz electrical stimuli applied with 100~A to the POA-SQ region, prolactin levels increased slightly by 30 min without reaching statistical significance (Fig. 2). Individual increases were observed in 3 out of 13 animals. Doubling current intensity induced a significant rise in plasma prolactin levels by 30 min, rapidly decreasing thereafter towards control levels (Fig. 2). Individual increases occurred in 13 out of 14 animals. Differences with the sham group were significant (P < 0.05),
1o Hz
0---0 100 uA, e---O 2OO uA, P O A - S O
4ra t (acute)
--"
150-
= 200uA,ARC-VMH
~'-"~ Sham
05)
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Z "J
//
50-
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o.
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I
I
I
0
30
60
90
Ti m e (rain)
Fig. 3. Effects of 10 Hz stimulation on plasma LH levels in male rats under pentobarbital anesthesia. Same as in Fig. 1.
o rat
Prolactin 10
Hz-
100 •
ac
o \\ ~..~,
increase
o no
change
,~'.-o ..~!
o qq o
-4
11
uA
9
""".,
7
5
3
1
Fig. 4. Brain electrode locations of the 10 Hz (100/~A) stimulated group, for the prolactin reported values. Schematic drawing at plane L 1.1 (modified from the De Groot atlasg). Suprachiasmatic (SQ) and arcuate nucleus (ARC) projected from plane L 0.6. fx, fornix; ac, anterior commissure; oq, optic chiasm.
o~rat
LH 10 Hz-lO0 •
ac 0
"~k,~
~
=
uA
increase
o no
change
-1 -2
-3 -4 -5
. . . . 11
. 9
7
5
.'1
..
3
1
Fig. 5. Brain electrode locations of the 10 Hz (100/~A) stimulated group, for the LH reported values. Same as in Fig. 4.
O•rat
lO Hz - 2 0 0 uA
(acute)
ARC-M
ED, EM.
150-
Prl
121---.t21 L H
T Ilc
E
lOO-
C
//
Z
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I-¢J
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E
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(8) (7)
\
/
II L___J 0.7 mm
/
Q.
I
I
I
i
o
30
60
90
Time
~minh/
Fig. 6. Plasma L H and prolactin levels in male rats after stimulation of the arcuate nucleus-median eminence region. Same as in Fig. 1.
319
Prolactin
o rat 10
Hz-200
ac 0 -1
\\
~,,z,,
"*.
-2
%
uA
•
increase
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no
change
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-3
-4 -5 .
11
.
.
.
9
7
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Fig. 7. Brain electrode locations of the 10 Hz (200/~A) stimulated group, for the prolactin reported values. Same as in Fig. 4.
and so were within group differences (P < 0.01). When a similar (200 #A) stimulation was applied to the VMH-ARC region, it failed to affect plasma prolactin levels (Figs. 2 and 7), except in 3 out of 15 animals. Stimulation delivered through closer electrode tips (interelectrode distance 0.7 mm) to the ARC-MED.EM. region at 10 Hz (200 #A) resulted in changes in plasma prolactin values (Fig. 6) with statistically significant differences between 0 vs. 30 min (P < 0.05) and 0 vs. 60 min (P < 0.05). Probably due to somehow large variances, ov~erall differences did not appear to be statistically significant. Individual increases were observed in 6 out of 7 animals. LH levels after 10 Hz (100 #A) stimulation were increased by 30 min, reaching significance by 60 min (P < 0.01), and were still high in the 90 min sample (P < 0.01) (Fig. 3). Significant increases from the sham group were present (P < 0.01). LH increases were observed in 12 out of 18 animals. Electrode locations mostly involved mid and ventral portions of the POA, ventral portions of the AHA and periventricularsuprachiasmatic region (Figs. 4 and 5). With 10 Hz stimulation delivered at 200/~A to the POA-SQ region, plasma LH levels rose significantly (Fig. 3). Values increased by 30 min and remained high throughout the experimental period. Although differences between 30, 60 and 90 min were not statistically significant, a notch in the response curve was apparent at 60 min, as was also the case in other experimental groups. Positive responses were obtained in 11 out of 15 cases. Additionally, 3 out of 5 animals showed an increase when the electrode pairs reached the junction between the AHA and VMH nuclei (Fig. 5). At variance with the prolactin response, LH levels increased after stimulation of the ARC-VMH region with 10 Hz (200/~A). Electrodes located in the region of the arcuate and basal ventromedial nuclei induced an increase in 14 out of 15 animals.
320
o
rat
L H 10
Hz-200
ac o
~ 5
~ increase
~I~
;, '",,,
- 1
•
11
•
.
9
no c h a n g e ,
•
uA
.
7
.fx
=
1
•
5
*
3
=l
,
1
Fig. 8. Brain electrode locations of the 10 Hz (200/~A) stimulated group, for the LI-I reported values. Same as in Fig. 4.
Differences with the sham group were significant (P < 0.01) and so were within group differences (P < 0.05). A similar response curve was obtained after stimulating the ARC-MED.EM. region (electrode tip separation 0.7 mm) with 10 Hz (200 #A) (Fig. 6). Overall differences were significant (P < 0.01). Stimulation with 200 Hz (100/~A) applied to the POA-SQ region induced similar increases in plasma LH and prolactin levels (Fig. 1). Prolactin differences with the sham group were significant (P < 0.01). LH levels were also significantly different from those of the sham group (P < 0.01). Stimulation with 200 Hz at 200/~A was discarded due to the high mortality rate. It was unclear whether this was due primarily to cardiac or respiratory failure. Stimulation with 3 or 10 Hz usually did not affect grossly the respiratory rhythm, which did occur at higher pulse frequency rates. DISCUSSION
In this work, positive results were obtained with frequencies of stimulation within the reported basal firing rates in the female rat. It can thus be presumed that neural elements involved in the control of hormonal (LH and prolactin) release in male rats are characterized by spontaneous basal discharge rates lower than the applied stimuli. Further, the data suggest a difference in this respect between prolactin and LH release controlling elements. Stimulation of the POA-SQ region at a rate of 3 Hz was effective in inducing a significant change in plasma prolactin levels, but not LH, by 30 min after stimulus onset. The biphasic pattern of prolactin release at this stimulation frequency could represent the true response to the applied stimuli or a combination of it (the early component), plus late 'unspecific' component probably related to experimental conditions. The latter is suggested by the hormonal behavior observed in other animal groups in which a tendency to give late high values was readily apparent, albeit
321 less marked. Changes in the level of anesthesia with time, plus accumulating effects of plasma/saline periodic exchange could be considered possible 'unspecific' factors capable of affecting hormonal levels. The responsiveness of prolactin to the 3 Hz stimulation suggests that its controlling elements have lower discharge rates than those controlling LH release. It should also be noted that prolactin maximum responses (average hormonal levels and number of responsive animals) were attained with 10 Hz stimulation. LH levels were maximally affected by the 50 Hz stimuli when applied to the POA-SQ region (Colombo, to be published). This stimulus frequency is close to the most effective frequency obtained by Jamieson and Fink 16 of 60 Hz. Yet, in the present work, the 10 Hz stimuli proved to be quite effective also in this respect. Differences between LH and prolactin response curve profiles were evident with l0 Hz stimuli, but tended to disappear with higher rates of stimulation. Thus, increasing stimulation frequencies tend to erase certain intrinsic differences between prolactin and LH controlling mechanisms. Stimulation of the VMH-ARC region with parameters fully effective when applied to the POA-SQ or ARC-MED.EM. regions did not result in prolactin increase, thus indicating a regional difference with the LH response. The average LH response consistently presented a notch at the 60 min sample, the nature of which remains to be established. In general, the average LH responses were quite similar after l0 Hz stimulation of either the POA-SQ, ARC-VMH or the ARC-MED.EM. regions. This was also the case after 50 Hz stimuli (Colombo, to be published), thus indicating a rather similar b~havior of neuronal elements in the considered region or, in fact, that they correspond essentially to the same set controlling LH release. It is pertinent to recall in this regard the POA and AHA projections to the hypothalamus as disclosed by autoradiographic methods after microinjections of tritiated amino acids 6-s,25. Those studies indicated the presence of POA and AHA axon terminals at the level of the arcuate nucleus and median eminence, with some AHA neurones projecting to the VMH nucleus. Special attention should be given to the fact that in no instance was an inhibitory effect on prolactin release observed after stimulation of the arcuate nucleus and median eminence. Careful consideration should be given to the experimental protocol, since pentobarbital is known to block ovulation12 as well as LH and prolactin surges during the afternoon of proestrus in female rats1, 29,al and to decrease LH levels in castrated rats ~. According to Wuttke and Meitesa0 pentobarbital reduces PIF hypothalamic content. Any increase in plasma prolactin levels due to stimulation of brain sites under these conditions would then have to be related to the release of PRF rather than on the inhibition of PIF. Yet, presumed PIF content in pentobarbital-treated hypothalami significantly changed between l0 min and 2 h. Thus, in searching for possible explanations of plasma prolactin increase under pentobarbital anesthesia one cannot completely discard an effect on PIF release. Stimulatory studies have so far failed to disclose inhibitory components for prolactin release within the arcuatemedian eminence region, except perhaps for the posterior region of the median eminence in sheep is. It could be reasonably presumed that stimulation applied to the
322 arcuate nucleus-median eminence area involves axon terminals containing the final messengers for pituitary function. If so, what characteristics turn PIF- (or dopamine-) containing terminals inexcitable or almost systematically inhibited (as judged from prolactin changes) although directly stimulated ? Is the PIF (or dopamine) pool rapidly depleted? Does POA-SQ region stimulation deplete those same terminals and inhibit P I F (or dopamine) synthesis? It has been proposed that pentobarbital affects both pre- and postsynaptic mechanisms3,20,21; thus, stimulation effects should also be analyzed in the chronic, awake animal. Interestingly, Gallo and Osland 13 reported inhibitory effects after median eminence stimulation on LH release in ovariectomized rats under chronic conditions, but not after estrogen treatment. Regarding possible mechanisms of functional segregation and frequency differentiation, the existence of patches of low safety factor at axon branching has been considered as a way of controlling two different effectors by one neuronal process in the invertebrate neuromuscular system 14,24,27. Operatively this would result in differential channelling according to the incoming frequency of spikes, thus controlling different effectors sensitive to a given bandwidth of spike frequencies. This remains as an attractive possibility yet to be described in the mammalian central nervous system with regard to neuroendocrine control mechanisms. ACKNOWLEDGEMENTS The author wishes to acknowledge the help of Prof. R. Leiva (Carrera del Personal de Apoyo of the C.O.N.I.C.E.T.) in statistical analysis, and Mrs. 1. Borzino for technical assistance. This work was supported by Grants 6846/74, 6419/74 and 7220/75 from the Consejo Nacional de Investigaciones Cientificas y T6cnicas (Argentina). The author is indebted to the Cfitedra de Anatomia, Facultad de Ciencias M6dicas, Universidad Nacional de Cuyo, Mendoza for permission to use electrophysiological equipment.
REFERENCES 1 Ajika, K., Krulich, L. and McCann, S. M., The effect of pentobarbital (Nembutal) on prolactin release in the rat, Proc. Soc. exp. Biol. (N. Y.), 141 (1972) 203-205. 2 Beattie, C. W., Campbell, C. S., Nequin, L. G., Soyka, L. F. and Schwartz, N. B., Barbiturate blockade of tonic LH secretion in the male and female rat, Endocrinology, 92 (1973) 1634-1638. 3 Blaustein, M. P., Barbiturates block calcium uptake by stimulated and potassium-depolarized rat sympathetic ganglia, J. Pharrnacol. exp. Ther., 197 (1976) 80-86. 4 Brownlee, K. A., In Statistical Theory and Methodology in Science and Engineering, J. Wiley and Sons, New York, 1965. 5 Bueno, J. and Pfaff, D. W., Single unit recording in hypothalamus and preoptic area of estrogentreated and untreated ovariectomized rats, Brain Research, 101 (1976) 67-78. 6 Conrad, L. C. A. and Pfaff, D. W., Axonal projections of medial preoptic and anterior hypothalamic neurons, Science, 190 (1975) 1112-1114. 7 Conrad, L. C. A. and Pfaff, D. W., Efferents from medial basal forebrain and hypothalamus in the rat. I. An autoradiographic study of the medial preoptic area, J. comp. NeuroL, 169 (1976) 185-220.
323 8 Conrad, L. C. A. and Pfaff, D. W., Efferents from medial basal forebrain and hypothalamus in the rat. II. An autoradiographic study of the anterior hypothalamus, J. comp. NeuroL, 169 (1976) 221-262. 9 De Groot, J., The rat forebrain in stereotaxic coordinates, Trans. roy. Neth. Acad. Sci., 52 (1959) 1--40. 10 Dyer, R. G. and Cross, B. A., Antidromic identification of units in the preoptic and anterior hypothalamic areas projecting directly to the ventromedial and arcuate nuclei, Brain Research, 43 (1972) 254-258. 11 Dyer, R. G., An electrophysiological dissection of the hypothalamic region which regulates the pre-ovulatory secretion of luteinizinghormone in the rat, J. Physiol. (Lond.), 234 (1973) 421-442. 12 Everett, J. W., Sawyer, C. H. and Markee, J. E., A neurogenic timing factor in control of the ovulatory discharge of luteinizing hormone in the cyclic rat, Endocrinology, 44 (1949) 234-250. 13 Gallo, R. V. and Osland, R. B., Electrical stimulation of the arcuate nucleus in ovariectomized rats inhibits episodic luteinizing hormone (LH) release but excites LH release after estrogen priming, Endocrinology, 99 (1976) 659-668. 14 Grossman, Y., Spira, M. E. and Parnas, I., Differential flow of information into branches of a single axon, Brain Research, 64 (1973) 379-386. 15 Hare, K. and Geohegan, W. A., Influence of frequency of stimulation upon response to hypothalamic stimulation, J. Neurophysiol., 4 (1941) 266-273. 16 Jamieson, M. G. and Fink, G., Parameters of electrical stimulation of the medial preoptic area for release of gonadotrophins in male rats, J. Endocr., 68 (1976) 57-70. 17 Lincoln, D. W. and Wakerley, J. B., Factors governing the periodic activation of supraoptic and paraventricular neurosecretory cells during suckling in the rat, J. Physiol. (Lond.), 250 (1975) 443461. 18 Malven, P. V., Immediate release of prolactin and biphasic effects on growth hormone release following electrical stimulation of the median eminence, Endocrinology, 97 (1975) 808-815. 19 Mogenson, G. J., Gentil, C. G. and Stevenson, J. A. F., Feeding and drinking elicited by low and high frequencies of hypothalamic stimulation, Brain Research, 33 (1971) 127-137. 20 Nicoll, R. A., Eccles, J. C., Oshima, T. and Rubia, F., Prolongation of hippocampal inhibitory postsynaptic potentials by barbiturates, Nature (Lond.), 258 (1975) 625-627. 21 Ransom, B. R. and Barker, J. L., Pentobarbital modulates transmitter effects on mouse spinal neurones grown in tissue culture, Nature (Lond.), 254 (1975) 703-705. 22 Renaud, L. P. and Martin, J. B., Electrophysiological studies on connections of hypothalamic ventromedial neurons in the rat: evidence for a role in neuroendocrineregulation, Brain Research, 93 (1975) 145-151. 23 Renaud, L. P., Influence of amygdala stimulation on the activity of identified tuberoinfundibular neurones in the rat hypothalamus, J. Physiol. (Lond.), 260 (1976) 237-252. 24 Spira, M. E., Yarom, Y. and Parnas, I., Modulation of spike frequency by regions of special axonal geometry and by synaptic inputs, J. Neurophysiol., 39 (1976) 882-899. 25 Swanson, L. W., An autoradiographic study of the efferent connections of the preoptic region in the rat, J. comp. NeuroL, 167 (1976) 227-256. 26 Terasawa, E. and Sawyer, C. H., Electrical and electrochemical stimulation of the hypothalamoadenohypophysial system with stainless steel electrodes, Endocrinology, 84 (1969) 918-925. 27 Waxman, J. G., Regional differentiation of the axon: a review with special reference to the complex of the multiplex neuron, Brain Research, 47 (1972) 269-288. 28 Whitehead, S.A. and Ruf, K.B., Responses of antidromically identified preoptic neurons in the rat neurotransmitters and to estrogen, Brain Research, 79 (1974) 185-198. 29 Wuttke, W. and Meites, J., Effects of ether and pentobarbital on serum prolactin and LH levels in proestrous rats, Proc. Soc. exp. Biol. ( N. Y.), 135 (1970) 648-652. 30 Wuttke, W., Gelato, M. and Meites, J., Effects of Na-pentobarbital on hypothalamic PIF, LRF and FSH-RF and on serum prolactin, LH and FSH. In Brain-Endocrine Interaction. Median Eminence: Structure and Function, S. Karger, Basel, 1972, pp. 267-279. 31 Yokoyama, A., Tomogane, H. and Ota, K., Prolactin surge on the afternoon of proestrus in the rat and its blockade by pentobarbitone, Experientia (Basel), 27 (1971) 578-579.
PREOPTIC-HYPOTHALAMIC
CONTROL
313
OF
LH
AND
PROLACTIN
RELEASE IN MALE RATS UNDER PENTOBARBITAL ANESTHESIA: DIFFERENTIAL RESPONSES TO ELECTRICAL STIMULATION
JORGE A. COLOMBO* Instituto de Investigaciones Cerebrales, Facultad de Ciencias Af~dicas, Universidad Nacional de Cuyo, Mendoza (Argentina}
(Accepted August 3rd, 1977)
SUMMARY In acute experiments under pentobarbital anesthesia, the brains of adult male rats were stimulated bilaterally with trains of monophasic pulses at different frequencies and current intensities through bipolar, concentric, stainless steel electrodes. Stimulation of the POA-SQ region at 3, l0 (200/~A) or 200 Hz (100/~A) increased plasma prolactin levels within 30 min of the onset of stimulation. Stimulus parameters fully effective when applied to the POA-SQ or ARC-MED. EM. regions were ineffective when applied to the ARC-VMH region. Maximal effects were obtained with 10 Hz (200/zA) stimuli applied to the POA-SQ region. LH levels were increased after stimulation of any of the three regions - - POASQ, ARC-VMH or ARC-MED. EM. Conversely to the prolactin response, LH levels did not rise after 3 Hz stimulation of the POA-SQ region. In male rats under pentobarbital anesthesia, LH levels seemed to be affected maximally with a stimulus frequency rate of about 50 Hz applied to the POA-SQ region. The results suggest a potential physiological role of frequency discrimination in the functional segregation of LH and prolactin release controls.
INTRODUCTION The preoptic-hypothalamic complex contains neural circuits controlling pituitary prolactin and luteinizing hormone (LH) release. Stimulation studies have been concerned primarily with the localization of circuits controlling the release of indivi* Member of the ResearchCareer of the ConsejoNacional de InvestigacionesCientificasy T6cnicas (Argentina). Present address: Department of Anatomy, College of Medicine, Medical Center,
Box 6, 12901 North 30th Street, Tampa, Fla. 33612, U.S.A.
314 dual hormones. The dense packing of neural elements suggests that several factors might be operating to assure a given set of outputs for a corresponding set of inputs. Among them, frequency response is a possible way to discriminate functions within a given neuronal population 15A9. A wide range of spontaneous firing frequency rates has been described in the rat for units located within the preoptic-hypothalamic complex5,1°,11,22,23,2s, yet they rarely exceed 10 spikes/sec. At least in the final common path of the neuroendocrine effector system information transfer appears to bear a direct relationship to changes in spike density17. The end result would thus depend essentially on changes in the number of potentials arriving at the final (effector) element during a given period. The existence of 'silent' and extremely slow firing units implies that a functionally significant increase in firing rates could be attained with what has traditionally been considered low frequency stimulation. Data presented in this paper yield evidence that elements controlling release of LH and prolactin within the preoptic-suprachiasmatic nucleus (POA-SQ), arcuate nucleus-ventromedial nucleus (ARC-VMH) and arcuate nucleus-median eminence (ARC-MED. EM.) regions do not share the same responses to a given set of applied electrical stimuli, and that they are sensitive to low frequency stimulation. Furthermore, data are consistent with the hypothesis that frequency discrimination is a possible mechanism for the separate control of release of the two hormones. MATERIAL AND METHODS Adult Holtzman male rats (250-330 g) maintained under controlled illumination (lights on 06.00-20.00 h) and fed with standard laboratory diet and water ad libitum were used. Animals were anesthetized with pentobarbital (35 mg/kg, i.p.). A tracheotomy was performed and a femoral artery cannulated for blood sample withdrawal. The animal was then placed in a stereotaxic instrument where it remained until the completion of the experiment. Animals were maintained warm with the aid of an incandescent bulb. A stainless steel, bipolar, concentric electrode pair was directed to preoptic or hypothalamic locations following the De Groot atlas 9 coordinates. Electrodes were made with 25 g tubing and 150/~m insulated inner wire, with similar characteristics to those used by Terasawa and Sawyer26. Bilateral electrical stimulation consisted of monophasic (outer barrel anode, inner wire cathode) rectangular pulses generated by an $4 Grass stimulator and driven through a variable resistor box placed in series with the preparation. Current intensity was continuously monitored through an oscilloscope and readjusted when needed. Standard parameters were: 0.5 msec width, 30 sec trains every other 30 sec, 100-200/zA current intensity, 3-10200 Hz, for a period of 30 min. Stimulation started 45 min after the electrode placement and not less than one hour after pentobarbital administration. After an initial (0 time) prestimulation sample (0.9 ml) was taken, blood sampling continued every 30 min during a total period of 90 min. Following blood centrifugation and plasma separation, blood cells were resuspended in physiological s~line ~nd injected back
315 after the next sample. At the end of the experiment electrode locations were established by means of the Prussian blue reaction after passing marking current through the electrode tips. Brains were perfused with K ferrocyanide and 10 ~ formalin and 80/~m frozen sections examined in a dissecting microscope. Plasma samples were stored frozen at --20 °C until radioimmunoassay procedures. L H and prolactin were measured in the same plasma sample by the double antibody radioimmunoassay technique, using the kits supplied by Dr. A. F. Parlow (NIAMDD, NIH). Results are expressed in ng/ml of plasma of the N I A M D D RatLH-RP 1 and N I A M D D Rat-Prolactin-RP 1. Analysis of variance 4 was used to compare differences between and within groups. RESULTS Sham-operated animals showed small, non-significant hormonal variations across time, with a tendency to give higher values by the end of the experimental period. Control (0 time) values of each experimental group did not differ significantly from those obtained in unstimulated controls. d t rat
150-
POA-SQ
(acute)
Prl I P - - 4 LH 0 - - - - 0
3Hz,200uA
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90
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Fig. 1. Bilateral stimulation of the POA-SQ region with 3 Hz (200/~A) or 200 Itz (100 ~A) upon LH and prolactin plasma levels in male rats under pentobarbital anesthesia. Mean 4- S.E. Figures in brackets = number of animals per group. S, period of stimulation.
316
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Fig. 2. Effects of l0 Hz stimulation on plasma prolactin levelsin male rats under pentobarbital anesthesia. Same asin Fig. 1. Stimulation of the POA-SQ region at 3 Hz (200 FA) affected prolactin but not LH plasma levels, except for the 90 min sample (Fig. 1). Prolactin values increased by 30 min, returned to control levels by 60 min and tended to give high values in the 90 min sample. Overall differences with the sham group were statistically significant (P < 0.01) and also at 30 min (P < 0.01) and 90 min (P < 0.01), and so were within group differences (P < 0.05), with 0 vs. 30 min P < 0.01 and 30 vs. 60 min P < 0.05. Individual increases (defined here as a 100~ increase above control level at 30 or 60 min) were observed in 6 out of 10 animals. On the contrary, L H levels remained practically unchanged (only 2 out of 12 animals produced a significant increase) except fora late increase by the 90 min sample (0 vs. 90 min P < 0.05) (Fig. 1). Most electrode placements were localized at midventral sites of the MPO and AHA, in some cases involving one of the suprachiasmatic nuclei. With l0 Hz electrical stimuli applied with 100~A to the POA-SQ region, prolactin levels increased slightly by 30 min without reaching statistical significance (Fig. 2). Individual increases were observed in 3 out of 13 animals. Doubling current intensity induced a significant rise in plasma prolactin levels by 30 min, rapidly decreasing thereafter towards control levels (Fig. 2). Individual increases occurred in 13 out of 14 animals. Differences with the sham group were significant (P < 0.05),
1o Hz
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Fig. 3. Effects of 10 Hz stimulation on plasma LH levels in male rats under pentobarbital anesthesia. Same as in Fig. 1.
o rat
Prolactin 10
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o~rat
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Fig. 5. Brain electrode locations of the 10 Hz (100/~A) stimulated group, for the LH reported values. Same as in Fig. 4.
O•rat
lO Hz - 2 0 0 uA
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Fig. 6. Plasma L H and prolactin levels in male rats after stimulation of the arcuate nucleus-median eminence region. Same as in Fig. 1.
319
Prolactin
o rat 10
Hz-200
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Fig. 7. Brain electrode locations of the 10 Hz (200/~A) stimulated group, for the prolactin reported values. Same as in Fig. 4.
and so were within group differences (P < 0.01). When a similar (200 #A) stimulation was applied to the VMH-ARC region, it failed to affect plasma prolactin levels (Figs. 2 and 7), except in 3 out of 15 animals. Stimulation delivered through closer electrode tips (interelectrode distance 0.7 mm) to the ARC-MED.EM. region at 10 Hz (200 #A) resulted in changes in plasma prolactin values (Fig. 6) with statistically significant differences between 0 vs. 30 min (P < 0.05) and 0 vs. 60 min (P < 0.05). Probably due to somehow large variances, ov~erall differences did not appear to be statistically significant. Individual increases were observed in 6 out of 7 animals. LH levels after 10 Hz (100 #A) stimulation were increased by 30 min, reaching significance by 60 min (P < 0.01), and were still high in the 90 min sample (P < 0.01) (Fig. 3). Significant increases from the sham group were present (P < 0.01). LH increases were observed in 12 out of 18 animals. Electrode locations mostly involved mid and ventral portions of the POA, ventral portions of the AHA and periventricularsuprachiasmatic region (Figs. 4 and 5). With 10 Hz stimulation delivered at 200/~A to the POA-SQ region, plasma LH levels rose significantly (Fig. 3). Values increased by 30 min and remained high throughout the experimental period. Although differences between 30, 60 and 90 min were not statistically significant, a notch in the response curve was apparent at 60 min, as was also the case in other experimental groups. Positive responses were obtained in 11 out of 15 cases. Additionally, 3 out of 5 animals showed an increase when the electrode pairs reached the junction between the AHA and VMH nuclei (Fig. 5). At variance with the prolactin response, LH levels increased after stimulation of the ARC-VMH region with 10 Hz (200/~A). Electrodes located in the region of the arcuate and basal ventromedial nuclei induced an increase in 14 out of 15 animals.
320
o
rat
L H 10
Hz-200
ac o
~ 5
~ increase
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Fig. 8. Brain electrode locations of the 10 Hz (200/~A) stimulated group, for the LI-I reported values. Same as in Fig. 4.
Differences with the sham group were significant (P < 0.01) and so were within group differences (P < 0.05). A similar response curve was obtained after stimulating the ARC-MED.EM. region (electrode tip separation 0.7 mm) with 10 Hz (200 #A) (Fig. 6). Overall differences were significant (P < 0.01). Stimulation with 200 Hz (100/~A) applied to the POA-SQ region induced similar increases in plasma LH and prolactin levels (Fig. 1). Prolactin differences with the sham group were significant (P < 0.01). LH levels were also significantly different from those of the sham group (P < 0.01). Stimulation with 200 Hz at 200/~A was discarded due to the high mortality rate. It was unclear whether this was due primarily to cardiac or respiratory failure. Stimulation with 3 or 10 Hz usually did not affect grossly the respiratory rhythm, which did occur at higher pulse frequency rates. DISCUSSION
In this work, positive results were obtained with frequencies of stimulation within the reported basal firing rates in the female rat. It can thus be presumed that neural elements involved in the control of hormonal (LH and prolactin) release in male rats are characterized by spontaneous basal discharge rates lower than the applied stimuli. Further, the data suggest a difference in this respect between prolactin and LH release controlling elements. Stimulation of the POA-SQ region at a rate of 3 Hz was effective in inducing a significant change in plasma prolactin levels, but not LH, by 30 min after stimulus onset. The biphasic pattern of prolactin release at this stimulation frequency could represent the true response to the applied stimuli or a combination of it (the early component), plus late 'unspecific' component probably related to experimental conditions. The latter is suggested by the hormonal behavior observed in other animal groups in which a tendency to give late high values was readily apparent, albeit
321 less marked. Changes in the level of anesthesia with time, plus accumulating effects of plasma/saline periodic exchange could be considered possible 'unspecific' factors capable of affecting hormonal levels. The responsiveness of prolactin to the 3 Hz stimulation suggests that its controlling elements have lower discharge rates than those controlling LH release. It should also be noted that prolactin maximum responses (average hormonal levels and number of responsive animals) were attained with 10 Hz stimulation. LH levels were maximally affected by the 50 Hz stimuli when applied to the POA-SQ region (Colombo, to be published). This stimulus frequency is close to the most effective frequency obtained by Jamieson and Fink 16 of 60 Hz. Yet, in the present work, the 10 Hz stimuli proved to be quite effective also in this respect. Differences between LH and prolactin response curve profiles were evident with l0 Hz stimuli, but tended to disappear with higher rates of stimulation. Thus, increasing stimulation frequencies tend to erase certain intrinsic differences between prolactin and LH controlling mechanisms. Stimulation of the VMH-ARC region with parameters fully effective when applied to the POA-SQ or ARC-MED.EM. regions did not result in prolactin increase, thus indicating a regional difference with the LH response. The average LH response consistently presented a notch at the 60 min sample, the nature of which remains to be established. In general, the average LH responses were quite similar after l0 Hz stimulation of either the POA-SQ, ARC-VMH or the ARC-MED.EM. regions. This was also the case after 50 Hz stimuli (Colombo, to be published), thus indicating a rather similar b~havior of neuronal elements in the considered region or, in fact, that they correspond essentially to the same set controlling LH release. It is pertinent to recall in this regard the POA and AHA projections to the hypothalamus as disclosed by autoradiographic methods after microinjections of tritiated amino acids 6-s,25. Those studies indicated the presence of POA and AHA axon terminals at the level of the arcuate nucleus and median eminence, with some AHA neurones projecting to the VMH nucleus. Special attention should be given to the fact that in no instance was an inhibitory effect on prolactin release observed after stimulation of the arcuate nucleus and median eminence. Careful consideration should be given to the experimental protocol, since pentobarbital is known to block ovulation12 as well as LH and prolactin surges during the afternoon of proestrus in female rats1, 29,al and to decrease LH levels in castrated rats ~. According to Wuttke and Meitesa0 pentobarbital reduces PIF hypothalamic content. Any increase in plasma prolactin levels due to stimulation of brain sites under these conditions would then have to be related to the release of PRF rather than on the inhibition of PIF. Yet, presumed PIF content in pentobarbital-treated hypothalami significantly changed between l0 min and 2 h. Thus, in searching for possible explanations of plasma prolactin increase under pentobarbital anesthesia one cannot completely discard an effect on PIF release. Stimulatory studies have so far failed to disclose inhibitory components for prolactin release within the arcuatemedian eminence region, except perhaps for the posterior region of the median eminence in sheep is. It could be reasonably presumed that stimulation applied to the
322 arcuate nucleus-median eminence area involves axon terminals containing the final messengers for pituitary function. If so, what characteristics turn PIF- (or dopamine-) containing terminals inexcitable or almost systematically inhibited (as judged from prolactin changes) although directly stimulated ? Is the PIF (or dopamine) pool rapidly depleted? Does POA-SQ region stimulation deplete those same terminals and inhibit P I F (or dopamine) synthesis? It has been proposed that pentobarbital affects both pre- and postsynaptic mechanisms3,20,21; thus, stimulation effects should also be analyzed in the chronic, awake animal. Interestingly, Gallo and Osland 13 reported inhibitory effects after median eminence stimulation on LH release in ovariectomized rats under chronic conditions, but not after estrogen treatment. Regarding possible mechanisms of functional segregation and frequency differentiation, the existence of patches of low safety factor at axon branching has been considered as a way of controlling two different effectors by one neuronal process in the invertebrate neuromuscular system 14,24,27. Operatively this would result in differential channelling according to the incoming frequency of spikes, thus controlling different effectors sensitive to a given bandwidth of spike frequencies. This remains as an attractive possibility yet to be described in the mammalian central nervous system with regard to neuroendocrine control mechanisms. ACKNOWLEDGEMENTS The author wishes to acknowledge the help of Prof. R. Leiva (Carrera del Personal de Apoyo of the C.O.N.I.C.E.T.) in statistical analysis, and Mrs. 1. Borzino for technical assistance. This work was supported by Grants 6846/74, 6419/74 and 7220/75 from the Consejo Nacional de Investigaciones Cientificas y T6cnicas (Argentina). The author is indebted to the Cfitedra de Anatomia, Facultad de Ciencias M6dicas, Universidad Nacional de Cuyo, Mendoza for permission to use electrophysiological equipment.
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