The milk ejection pathway in brain studied with the 2-deoxyglucose method

Brain Research, 273 (1983) 291-296 Elsevier

291

The Milk Ejection Pathway in Brain Studied with the 2-Deoxyglucose Method RUTH C. SUTHERLAND* and GEORGE FINK

M R C Brain Metabolism Unit, University Department of Pharmacology, 1 George Square, Edinburgh EH8 9JZ ( U. K.) (Accepted January llth, 1983)

Key words: milk ejection - - 2-deoxyglucose- - ascending pathway - - paraventricular and supraoptic nuclei - - pituitary gland

The neural pathways involved in the milk ejection reflex have been studied with the aid of the 2-deoxyglucose (2DG) method. All the experiments were carried out on Wistar female rats, 9-11 days post-partum, which had been separated from their pups (except for one) overnight. The effect of suckling on the relative metabolic activity (RMA) of the brain was studied in conscious rats and in rats anaesthetized with urethane. Control animals were similarly treated but were not suckled. In addition, the effect of mammary nerve stimulation on RMA was studied in animals anaesthetized with urethane; sham-operated animals served as controls for this group. Suckling (minimum of 10 pups) in conscious animals had no apparent effect on the RMA of anyof the brain areas measured. However, in anaesthetized rats, suckling produced a significant increase in the RMA of the paraventricular and supraoptic nuclei (PVN and SON), but had no effect on the RMA of any other brain area or the pituitary gland. Stimulation of the mammary nerve, with a stimulus that causes milk ejection and an increase in prolactin release, produced a significant increase in the RMA of the PVN, SON, the pars distalis and pars nervosa and the spinothalamic tract, and a significant decrease in the ventromedial and mediodorsal nuclei of the thalamus, the zona incerta, the red nucleus and the ventral nucleus of the lateral lemniscus. These results show that suckling significantly increases the metabolic activity of afferent terminals in the PVN and SON. Activation of the cell bodies of the PVN and SON, as assessed by increased RMA of the pituitary gland, could be evoked by the more intense stimulus of mammary nerve stimulation. The ascending pathway from the mammary nerve involves the spinothalamic tract but could not be traced beyond the midbrain. The lack of effect of suckling in conscious animals may have been due to the inhibitory influence of stress mediated by forebrain structures.

INTRODUCTION

cided with the spinothalamic tract. U r b a n et al.18 were not able to confirm this in the rabbit; they found

Milk ejection is produced by a n e u r o h o r m o n a l reflex. Suckling generates a neural signal which is conveyed by the m a m m a r y nerve and an ascending pathway to the forebrain where, by activating the magnocellular n e u r o n e s of the paraventricular and supraoptic nuclei (PVN and SON), the signal triggers the release of oxytocin 2. Activation of the magnocellular n e u r o n e s is k n o w n to consist of a brief (2-6 s) accelerated discharge, which occurs 10-12 s before milk ejectiont3, t9, but neither the anatomy of the ascending pathway nor the mechanism by which it activates the magnocellular neurones are understood. Several workers4,8,15-t7 using electrical stimulation and surgical lesions concluded that in the guinea pig, rabbit and goat the afferent pathway of the midbrain was discrete and coin-

that in the midbrain the pathway was diffuse and scattered across the medial and lateral t e g m e n t u m . Surgical lesions of the forebrain suggested that a central inhibitory mechanism for milk ejection is normally present. This inhibition which appears to be mild in the rat, could be blocked by section of the septo-hippocampal pathwayt4. The aim of the present study was to determine whether the 2-deoxyglucose ( 2 D G ) method for measuring metabolically active pathways in the braint2 could be used to answer some of these questions. In addition to using the natural stimulus of suckling in conscious and anaesthetized animals, we also studied the effect of m a m m a r y nerve stimulation which is an effective stimulus for causing milk ejection in the anaesthetized rat 9.

* Ethel and Colin Gordon Scholar to whom reprint requests should be sent. 0006-8993/83/$03.00© 1983 Elsevier Science Publishers B.V.

292 MATERIALS AND METHODS

Significance of differences was assessed using the unpaired t-test.

Animals The animals used were lactating rats of the Wistar strain, 330-415 g body weight, supplied by Charles River (U.K.), with litters of 10-15 pups. The animals were housed in individual cages under conditions of controlled lighting (14 h light-10 h dark) and temperature (22 °C) and were allowed free access to diet 41B and tap water. Experiments were carried out on days 9-11 post-partum after overnight separation of the young from the doe, apart from 1 pup from each litter which was left with the doe.

Autoradiography with [14C]2-deoxyglucose 2-Deoxyglucose (Amersham International; spec. act. 60 mCi/mmol in 3% ethanol) was injected intravenously at a dose of 150/,Ci/kg body weight (0.5 ml final volume with sterile 0.9% saline solution). After 45 min the rats were killed by decapitation and the brain and pituitary gland removed en-bloc. The tissue was then frozen in 2-methylbutane cooled to ----45 to --55 °C in dry-ice and mounted in Tissue Tek II (Miles, IL). Serial 20/zm coronal sections were cut immediately on a cryostat at - - 2 2 °C. The sections were dried rapidly on a hot plate at 60 °C and exposed to Kodak SB-5 X-ray film for 7 days at - - 1 5 °C. To permit quantification, a series of [14C]methyl methacrylate standards precalibrated for 20/~m tissue sections (concentration range 50-1057 nCi/g) were processed with each autoradiograph. The optical density (OD) of selected areas was measured using a Quantimet 800 (Cambridge Instruments) and a mean O D for each structure obtained from a minimum of 10 determinations over 5 sections. The [14C]concentration for each structure was then computed using the tissuematched standards, and was expressed as a ratio relative to the [~4C]-concentration of the corpus callosum (relative metabolic activity, RMA). Throughout the study the ratio of [14C] in the corpus callosum and internal capsule remained constant within and between individual animals and experimental groups. Therefore, it was justified to assume that areas of white matter quite distinct topographically and in terms of blood supply, are stable in terms of 2DG uptake and OD, and so to use the corpus callosum as an internal standard.

Experimental design The following groups of animals were studied. (i) Conscious animals. The pups (minimum of 10) were replaced and allowed to suckle for 20 min before 2DG was administered by way of an intra-atrial cannula that had been inserted according to the method of Greig3 24 h previously under halothane anaesthesia. (ii) Anaesthetized animals. Does were injected with urethane (ethyl carbamate) i.p. at a dose of 1 g/kg body weight, and the external jugular vein exposed. After 3 h, during which the does were left undisturbed under a warming lamp, the pups (minimum of 10) were replaced and allowed to suckle for 1 h before the i.v. injection of 2DG. This regime was based on that of Burnet and Wakerley 1 who found a good prolactin response to suckling in animals anaesthetized with urethane and which were allowed to remain undisturbed 3 h after surgery and before replacement of the pups. (iii) Mammary nerve stimulation. Animals were anaesthetized with urethane and a mammary nerve exposed according to the method of Mena et al. 9. The external jugular vein was also exposed. After a 45-60 min rest period 2DG was injected intravenously and the nerve stimulated using a Digitimer Neurolog system. The parameters of stimulation used were pulses of 1 ms, 10 V and 20 Hz for 10 s followed by 10 s of rest. Sham-operated, control animals, were subjected to a similar procedure but the mammary nerve was not stimulated. RESULTS Table I shows that in the conscious suckled animals there was no significant difference in the R M A of any of the areas measured compared with control unsuckled animals. The anaesthetized suckled animals, however, showed a significant increase in R M A of the supraoptic (SON) and paraventricular (PVN) nuclei (P < 0.005) above control anaesthetized unsuckled animals (Table I). No significant change in RMA occurred in the pars nervosa (PN) or pars distalis (PD) or in any of the mid- and hindbrain structures measured in these rats. Table I and Figs. 1-3

~

~

~.~ ~

~,~

....

~

,-~

.

_

" ~ - ~ ~.

~

~.~ ,,=, ~ ~ -

~.

~

- 6 ~ ~"

A ~'.~ ~ .~'A

~ ~.~.~

o

"~ -.

5 4

M a m m a r y nerve stimulation Sham control

~~

1.16±0.05

4

1.89+0.17" 1.41+0.03

1.31+0.07

1.62+0.03"***

1.16+0.03 1.14+0.07

PVN

0.70±0.08

0.76+0.08

0.86+0.09 0.64+0.03

Pars distalis

1.68+0.12"** 0.83+0.04* 1.11+0.09 0.66+0.04

1.33+0.18

1.62±0.20

1.09±0.05 1.13+0.17

Pars nerv osa

1.25+0.01"* 1.17+0.03

1.12+0.06

1.10±0.06

1.10±0.04 1.05+0.03

TSTH

2.14+0.07" 2.48±0.12

2.00+0.18

1.94±0.08

2.57+0.26 2.42±0.24

VMT

2.02+0.09* 2.42+0.19

1.90+0.18

1.89+0.11

2.56+0.20 2.32+0.18

MDT

1.80+0.03" 1.99+0.07

1.76+0.02

1.74+0.06

1.99+0.19 2.03+0.13

ZI

~

o

~-~

~~.~, ~,

~ .

~

°o=~

~ = ~, ~ o .

:ii ii ¸ i:~i'~ml

;ignificantly different from corresponding control group: * P < 0.05; ** P < 0.02; *** P < 0.01; **** P < 0.005.

1.69+0.11"* 1.25+0.07

1.53+0.04"***

1.08+0.02 0.92+0.11

5

4 4

~uckling 2ontrol

SON

•naesthetised and suckling 3ontrol anaesthetised

No. o f animals

Freatment

1.87+0.04" 2.20+0.14

1.81+0.21

1.59±0.03

2.02±0.15 1.99+0.10

NR

~%~?i~ ¸

2.16+0.07"*** 2.87+0.15

2.24+0.24

2.21+0.10

2.36+0.10 2.23+0.06

LLV

Values are expressed as a ratio ( m e a n s ± S . E . M . ) of [14C]-concentration of the selected region to [~4C]-concentration of the corpus callosum. S O N --- supraoptic nuclei; P V N = paraventricular nucleus; T S T H = spinothalamic tract; V M T = ventromedial thalamic nucleus; M D T = mediodorsal thalamic nucleus; ZI = zona incerta; N R = red nucleus; LLV = ventral nucleus of the lateral lemniscus.

Local [t4C]-concentrations o f selected brain regions and the pituitary gland in rats

TABLE I

tO

294 internal capsule, substantia nigra, habenular nucleus, lateral tegmentum, reticular formation, raphe nucleus and periaqueductai grey. In none of these areas was there a significant difference in RMA between experimental and control groups. DISCUSSION

Fig. 2. Autoradiographs of coronal sections of the brain at the level of the paraventricular nucleus (PVN; arrow) in rats injected with [14C]2-deoxyglucoseand the mammarynerve stimulated: PVN in sham-operated (a) and mammary nerve-stimulated animals (b). RMA in some brain areas compared with that in sham-operated animals. Decreases in RMA occurred in the ventromedial and mediodorsal thalamic nuclei, zona incerta and red nucleus (P < 0.05) and in the ventral nucleus of the lateral lemniscus (P < 0.005). In addition to the brain and pituitary areas shown in Table I, the RMA of the following brain areas was determined; suprachiasmatic nuclei, medial forebrain bundle, amygdala, ventral thalamic nucleus,

An increase in 2DG uptake is known to reflect an increase in metabolism 12, notably that associated with sodium pump activity7. The lack of an increase in RMA in the hypothalamo-neurohypophysial system in the conscious suckled rat is therefore surprising since substantial transient increases in activity of the magnocellular neurones have been shown to occur in response to suckling in the conscious rat 13. Slow wave sleep is necessary for milk ejection 5 and this may have been prevented by stress in these animals, thus inhibiting the milk ejection reflex. In order to minimize the effects of external stimuli on the doe, and to promote a period of slow wave sleep, an anaesthetized suckled group was therefore included in the study. Anaesthesia itself does not abolish the milk ejection reflex 6. In these animals the RMA of the SON and PVN increased significantly above control values but no change in RMA was found in any mid- and hindbrain structures measured, or in the pituitary gland. Mammary nerve stimulation resulted in an increase in RMA of the PVN, SON, PN and PD. This is consistent with the fact that similar parameters of mammary nerve stimulation produced an increase in intra-mammary pressure indicative of oxytocin release 9, and an increase in plasma prolactin concentrationS0. Whether the increased RMA in the hypothalamo-neurohypophysial system reflects only terminal activity, as suggested by Schwartz et al. t~ or whether activity in the cell bodies is also increased cannot be resolved using this method of autoradiography due to the limits of resolution. The anatomical distribution of the increase in the RMA of the PVN and SON was precisely located in the area of the cell bodies of these nuclei, and this is consistent with a possible increase in the metabolism of the cell bodies as well as of the nerve terminals of afferent projections to the PVN and SON. However, the increase in 2DG uptake by the PVN and SON in the anaesthetized suckled group with no concomitant increase in RMA of the pituitary suggests that we mea-

295

II

Ilmf

b Fig. 3. Autoradiographs of coronal sections of the pituitary gland in rats injected with [ 14C]2-deoxyglucose showing the pars nervosa and pars distalis in sham-operated (a) and mammary nerve-stimulated animals (b).

296 sured terminal activity, and that the magnocellular neurones themselves were not activated. Activation

fuse 18 afferent pathway. We found no increase in

of these neurones, sufficient to be detected by the

or medial forebrain bundle suggesting that perhaps

2DG method, did occur after m a m m a r y nerve stimulation which presumably is a more intense stimulus

the pathway is too diffuse for any increased activity to be detected by the 2 D G method. The central in-

than suckling. The R M A of the spinothalamic tract was also increased in m a m m a r y nerve-stimulated an-

hibitory mechanism is thought to be associated with the septohippocampal pathwayt4 but we found no

imals, and this is consistent whith the ascending milk ejection reflex path r u n n i n g coincident with that tract 4,s,15-17. Electrophysiological investigations in

experimental groups. The reason for the decrease in R M A of the thalamic and red nuclei, the ventral nu-

the rabbitlT, TM have provided conflicting evidence for

cleus of the lateral lemniscus and the zona incerta in

the precise anatomical location of the milk ejection

the animals in which the m a m m a r y nerve was stimulated is not clear, but this decrease may reflect inhibition of meelated to the central effects of peripheral nerve stimulation.

path through the midbrain. The activation of a central inhibitory mechanism that overrides the milk reflex has been suggested 1~.15 as a possible explanation

R M A of the lateral t e g m e n t u m , reticular formation

change in R M A of these structures in the different

for the conflicting data for a discrete 17 versus a dif-

REFERENCES 1 Burnet, F. R. and Wakerly, J. B., Plasma concentrations of prolactin and thyrotrophin during suckling in urethaneanaesthetized rats, J. Endocr., 70 (1976) 429--437. 2 Cross, B. A., Dyball, R. E. J., Dyer, R. G., Jones, C. W., Lincoln, D. W., Morris, J. F. and Pickering, B. T., Endocrine neurones, Rec. Progr. Horm. Res., 31 (1975) 243-286. 3 Greig, F., Serial luteinizing hormone measurement in cannulated unanaesthetised rats, J. Physiol. (Lond.), 218 (1971) 29P-30P. 4 Knaggs, G. S., McNeilley, A. S. and Tindal, J. S., The afferent pathway of the milk-ejection reflex in the mid-brain of the goat, J. Endocr., 52 (1972) 333-341. 5 Lincoln, D. W., Correlation of unit activity in the hypothalamus with EEG patterns associated with the sleep cycle, Exp. Neurol., 24 (1969) 1-18. 6 Lincoln, D. W., Hill, A. and Wakerley, J. B., The milkejection reflex of the rat: an intermittent function not abolished by surgical levels of anaesthesia, J. Endocr., 57 (1973) 459-476. 7 Mata, M., Fink, D. J., Gainer, H., Smith, C. B., Davidsen, L.. Savaki, H., Schwartz, W. J. and Sokoioff, L., Activity dependent energy metabolism in rat posterior pituitary primarily reflects sodium pump activity, J. Neurochem., 34 (1980) 213-215. 8 Mena, F. and Beyer, C., Effect of spinal cord lesions on milk ejection in the rabbit, Endocrinology, 83 (1968) 615-617. 9 Mena, F., Pacheco, P., Aguayo, D., Clapp, C. and Grosvenor, C. E., A rise in intramammary pressure follows electrical stimulation of mammary nerve in anaesthetized rats, Endocrinology, 103 (1978) 1929-1936. 10 Mena, F., Pacheco, P. and Grosnevor, C. E., Effect of electrical stimulation of mammary nerve upon pituitary and plasma prolactin concentrations in anaesthetized lactating

rats, Endocrinology, 106 (1980) 458--462. 11 Schwartz, W. J., Smith, C. B., Davidsen, L., Savaki, H., Sokoloff, L., Mata, M., Fink, D. J. and Gainer, H., Metabolic mapping of functional activity in the hypothalamoneurohypophyseal system of the rat, Science, 205 (1979) 723-725. 12 Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, M. H., Patlak, C. S., Pettigrew, K. D., Sakurada, O. and Shinohara, M., The [14C]deoxyglucose method for the measurement of local cerebral glucose utilisation: theory, procedure and normal values in the conscious and anaesthetized albino rat, J. Neurochem., 28 (1977) 897-916. 13 Summerlee, A. J. S. and Lincoln, D; W., Electrophysiological recordings from oxytocinergic neurones during suckling in the unanaesthetized lactating rat, J. Endocr., 90 (1981) 255--265. 14 Tindal, J. S. and Blake, L. A., A neural basis for central inhibition of milk ejection in the rabbit, J. Endocr., 86 (1980) 525-531. 15 Tindal, J. S. and Knaggs, G. S., Further studies on the afferent path of the milk-ejection reflex in the brain stem of the rabbit, J. Endocr., 66 (1975) 107-113. 16 Tindal, J. S., Knaggs, G. S. and Turvey, A., The afferent path of the milk-ejection reflex in the brain of the guinea pig, J. Endocr., 38 (1967) 337-349. 17 Tindal, J. S., Knaggs, G. S, and Turvey, A., The afferent path of the milk-ejection reflex in the brain of the rabbit, J. Endocr., 43 (1969) 663--671. 18 Urban, I., Moss, R. L. and Cross, B. A., Problems in electrical stimulation of afferent pathways for oxytocin release, J. Endocr., 51 (1971) 347-358. 19 Wakerley, J. B. and Lincoln, D. W., The milk ejection reflex of the rat: a 20- to 40-fold acceleration in the firing of paraventricular neurones during oxytocin release, J. Endocr., 57 (1973) 477-493.