adjacent hypothalamic and limbic areas terminate on neurons in the LPA-LH-MFB neuropil. Horseradish peroxidase. Hypothalamic interconnections.
Lateral Preoptic Area and Lateral Hypothalamic Interconnections Demonstrated by Horseradish Peroxidasely2 M. J. WAYNER,”
Brain Research Laboratory,
F. C. BARONE,
Syracuse
S. L. SCHAROUN
University, 601 University Avenue,
Syracuse, NY 13210
AND R. GUEVARA-AGUILAR
Department0
AND H. U. AGUILAR-BATURONI
de Fisiologia, Facultad de Medicina, Universidad National Apartado Postal 70250, Mexico 20, D. F.
WAYNER, M. J., F. C. BARONE, S. L. SCHAROUN,
Autonoma
de Mexico
AND H. U. AGUILAR-BATURONI. peroxidase. BRAIN RES. peroxidase, 30% Sigma Type VI, was administered iontophoretically to the lateral preoptic area (LPA) and lateral hypothalamus (LH) of male hooded rats. Animals were perfnsed intracardially on
Laterul preoptic area and lateral hypothalamic BULL. 5: Suppl. 4, 181-188, l%O.-Horseradish
R. GUEVA~-AGUILAR
interconnections
demonstrated
by horseradish
the following day, brains were removed and later sliced into 3&50 pm sections and processed with DAB and BDH for the brown and blue reaction products. Both LPA and LH ejections of horseradish peroxidase (HRP) resulted in the extensive labeling of soma and axons along the entire extent of the LPA, LH and the medial forebrain bundle (MFB). In addition, ejections in both areas resulted in similar labeling in the nucleus accumbens, stria hypothalamic tract, stria terminalis, medial hypothalamic area, amygdaloid nuclei, and the zona incerta. These results demonstrate a considerable degree of interconnectivity within the LPA and LH along the entire extent of the MFB and that presynaptic inputs from other adjacent hypothalamic and limbic areas terminate on neurons in the LPA-LH-MFB neuropil. Horseradish peroxidase Medial forebrain bundle
Lateral hy~thal~us Hypothalamic interconnections Stomach distension Vagus nerve stimulation
THE lateral preoptic area (LPA) and lateral hypothalamus (LH) are implicated in the neurology of ingestive behavior. Both the LPA and LH contain cells which are affected by osmotic stimulation [4, 6, 7, 20, 271 and severe deficits in ingestive behavior occur following the destruction of these areas [ 1, 2, 9, 22, 261. The significance of the LPA-LH area in the control of spinal motor excitability and behavior has been discussed [24,25]. Sensory inputs from the entire oropharyngeal region and gastrointestinal tract are required for normal ingestive behavior. Activity of cells in the LPA-LH region are highly correlated with changes in the physiologic~ conditions which precede and accompany drinking f4, 5, 17, 271. Neurons in the LPA-LH area are particularly sensitive to gastric water infusion and distension within a time period during which changes in drinking would normally occur [4]. Cells observed in 24-hr water deprived animals were more sensitive to water infusions and less sensitive to stomach distension when compared to cells recorded in animals maintained on ad lib eating and drinking.
Lateral preoptic area
Neurons in this region are also affected by electrical stimulation of the cervical vagus nerve which might be related to normal gastrointestinal atTerent activation [3,5]. Consistent with this hypothesis is the fact that LPA-LH cells tested and affected by gastric distension are affected similarly by vagus nerve stimulation [5]. Resection of the gastric vagal nerves eliminated distension induced alteration of hypothalamic activity [3]. Figure 1 is an illustration of the activity of a cell located in the LPA. Mean spikes per set for 30 set periods are presented as a function of time in min. Standard errors of the mean are also indicated for each 30 set period. Room temperature water was infused into a gastric balloon at a rate of I. 15 mlimin for 10 min for a total of 11.5 ml of water. Arrows indicate when the gastric water balloon fiiing began and ended and when the balloon was emptied during the expetiment. The cell appeared to respond to both changing distension of the stomach and a maintained stretch of the stomach wall. This interpretation is substantiated by the fact that as
‘Supported in part by a grant from the NINCDS USPHS No. 13543. ‘The authors would like to express their appreciation to Dr. W. J. H. Nauta and his colleagues in his laboratory at MIT, Cambridge, MA for assistance in the utilization of the HRP technique. “Reprint requests to Dr. M. J. Wayner, Brain Research Laboratory, 601 University Avenue, Syracuse, NY 13210.
Copyright 0 1980 ANKHO
International Inc .-0197-4580/80/010181-05$01.~/0
IX?
WAYN F‘K 1:7 .A!. STOP FllLlNG
EMPTY
2*23. :220 v :
21-
:20: 19Y z * 18f Y 173! 16 i:
0'
-3
-2
-1
I
0
I
1
2
3
4
5
TIME
IN
6
7
8
9
IO
11
12
13
14
IS
MlN!JlES
FIG. I. Mean spikes per set for 30 set periods (5S.E.) presented as a function of time in min for an WA neuron. Arrows indicate when the gastric water balloon fdling and emptying started and stopped and when the gastric balloon was emptied during the experiment. Room temperature tap water was infused at a rate of I. I5 ml/mitt.
the balloon was filled, mean discharge frequency and variability increased but when the balloon filing ceased, only variability remained elevated and mean discharge frequency returned to pre-distension baseline. In addition, when the gastric balloon was emptied an immediate decrease in discharge frequency occurred with a concomitant decrease in variability. A later stable mean discharge frequency was observed. Figure 2 is another illustration of the activity of the same teuron. Mean spikes per set for 10 set periods are
-
5v
a u
IOV
-,
-2-o
15v 2ov
-io
TIME
IN
0
i0
20
SECONDS
FIG. 2. Same neuron as in Fig. I. Mean spikes per set for IO set periods (2 S.E.) presented as a function of time for 20 set before and 30 set after 4 set of vagus nerve stimulation. Vagus nerve stimulation occurred just prior to 0 sec. Frequency was held constant at 100 Hz and 5 V (solid circles), IO V (triangles), ISV (squares), and 20 V (open circles) were applied to the nerve.
presented as a function of time for 20 set before and 30 set after 4 set of vagus nerve stimulation. Standard errors of the mean are also indicated for each 10 set period. Trains of 0.5 msec square waves were administered to the nerve at 100 Hz just prior to 0 sec. A clear voltage related increase in discharge frequency can be observed as stimulation voltages were increased from 5 V (solid circles), to 10 V (triangles), to I5 V (squares), to 20 V (open circles). Because LPA and LH neurons are affected in a similar manner by visceral [4,5] and osmotic [23,27] stimulation associated with drinking, the possibility of functionahy significant interconnections exists. Neurologically, the LPALH-MFB region of the hypothalamus is characterized by long MFB fibers and a series of neuronal loops interconnecting the dendrites of path neurons [ 131. Rostral path neurons receive a unique contribution from the medial preoptic and anterior hypothalamic nuclei whereas progressively caudal path neurons receive their primary supply from the ventromedial and premammillary nuclei 113, 14, 151. In general, the lateral preoptic-lateral hypothalamic region has a common afferent source from the medial forebrain longitudinal fiber system with considerable additional input to the rosttal portion from the amygdala, pyriform cortex and olfactory tubercle, and a large afferent supply to the caudal regions from the ascending reticular formation. Although there appear to be reciprocal connections from the LPA to the septal nuclei, olfactory tubercle. anterior olfactory nucleus and amygdala, the major efflux of fibers from path neurons and other medial forebrain sources seems to be into the mesencephahc tegmentum where they make extensive contacts with reticular neurons and form a structural intermediary connecting forebrain-hypothalamic regions with autonomic and somatic motor systems in the lower brain stem and spinal cord [12, 13, 14,21, 28). A direct inhibitory input between the LPA and LH related to drinking has also been suggested 1231.
LPA AND LH INTERCONNECTIONS
183
WAYNER L7 AL..
IX4
Since there is little, if any, anatomical data which illustrate the direct connections between neurons of the LPA an LH. the present study was conducted. Specifically, the purpose of the present investigation was to eject horseradish peroxidase by means of microiontophoresis into the LPA and LH and to determine retrograde transport and identification of interconnections. The most extensive labeling of soma and axons were observed along the LPA-LH-MFB following both LPA and LH horseradish peroxidase ejections which indicates considerable interconnectivity between the L-PA and LH along the MFB.
METHOD
Aninwls
Male Long-Evans rats, 300400 g in weight, were selected from our colony. Animals were housed in individual cages, ate standard Purina lab chow and drank water ad lib, and were kept on a constant light-dark cycle. The 12 hr light phase began at 0600 and was followed by a 12 hr dark phase. Room temperature was maintained at 70 t 2” F.
Rats were anesthetized with 3 cc/kg of Equi-Thesin (Jensen-SaJsbery Laboratories) and fixed in a stereotaxic instrument with the skull horizontal, lambda and bregma in the same horizontal plane. A longitudinal incision was made in the scalp and the skull was exposed. Predetermined holes were drilled in the skull over the hypothalamus according to a rat brain atlas I1 1I. The meninges were removed and the exposed brain tissue was covered with isotonic saline. lontophoretic ejections of horseradish peroxidase (HRP) were made in the LPA-MFB and mid LH-MFB. Ejection placements in the LPA-MFB were at the following coordinates, in mm, with the anterior coordinate relative to bregma, the lateral coordinate relative to the sagittaJ suture, and the ventral position measured from the surface of the brain: A = + I .2, L= + 1.5, V=S.O. Ejection placements in the LH-MFB were at the following coordinates: A= - 1 .O, L= +2.0, V=8.5. The iontophoretic ejections of HRP, Sigma Type VI, were made by passing 1.5-3.0 PA anodal current for 8-12 min through a glass microelectrode (Frederick Haer and Co.) which had been pulled, broken to a tip diameter of 30-40 pm, and filled with a 30% solution of HRP [lo]. After a 20-24 hr survival period, rats were perfused intracardially with 300 ml of 20% sucrose in phosphate buffer @H=7.4), followed by 300 ml of 1.25% glutaraldehyde plus 1% paraformaldehyde in phosphate buffer (pH=7.4). Brains were removed and immediately immersed in 20% sucrose in phosphate buffer (pH=7.4) and stored overnight at 4°C. The following day brains were sliced on a freezing microtome into 30-50 pm sections in the frontal plane. Some sections were processed with DAB tetra HCl for the brown reaction product according to the Nauta technique and lightly counterstained with cresyl violet. Other sections were processed with BDH for the blue reaction product according to the Nauta technique and lightly counterstained with neutral red [ 161. Both light and dark field microscopy were utilized in the identification and analysis of labeled axons and cell bodies. The exact locations of labeled soma were determined on drawings made from the individual sections in conjunction with plates from the Konig and Klippel rat brain atlas [ 111.
FIG. 4. Dark field photornicmgraph of a representative & ejection site in the LPA. Section was processed with DAB for the brown reaction and counterstained with cresyl violet. All ejection sites used in the analysis were approximately ZOOpm and loc’:titedwithin the I.PA-MFB area. RISUL’I 5
Successful ejection sites, approxtmately 500 pm in dnuneter. were identified in the LPA-MFB area of 4 animals and in the LH-MFB area of 4 other animals. The data from these 8 animals were used in the present description of the results. Data collected from a representative LPA HRP ejection is illustrated in Fig. 3 which demonstrates the retrograde transport of HRP from the LPA to the LH. The center of the ejection site is indicated by a star in the LPA-MFB area on plate 19. Figure 4 is a dark field photomicrograph illustrating the center of the HRP ejection site in the LPA of a representative animal. Ejection site diameter was approximately So0 pm located within the LPA-MFB area. Labeled neurons and axons in Fig. 3 are depicted by solid circles and wavy lines respectively. Each circle represents the location of approximately 3 contiguous identified soma. Anterior and dorsal movement of HRP from the injection site was observed. Neurons and axons were identified in the stria hypothalamic tract and stria terminalis. nucleus accumbens, and anterior MFB. Medial HRP movement was also indicated and labeled neurons were observed in the medial preoptic area. Posterior movement was observed along the posterior LPA-MFB.
185
LPA AND LH INTERCONNECTIONS
--
WA’t’NER k7 .A1
186
FIG. 6. Dark field photomicrograph of a representative HRP ejection site in the mid LH. Section was processed with BDH for the blue reaction and counterstained with neutral red. All ejection sites
used in the analysis were approximately 500 pm and located within
FIG. 7. Light field photomicrograph of a labeled cell located m the LPA. HRP was transported retrogradely to soma from the ejection site located in the mid LH-MFB area. Section was processed with BDH for the blue reaction and counterztained with neutral red
the LH-MFB.
throughout the LH-MFB area, amygdaloid nuclei, zona incerta, and medial hypothalamic areas. The most extensive labeling of axons and soma were observed along the LPALH-MFB extending to the most posterior portion of the MFB. Data from a representative LH HRP ejection is illustrated in Fig. 5 which demonstrates the retrograde transport of HRP from the LH to the LPA and is presented in a manner similar to that in Fig. 3. The center of the ejection site is indicated by a star on plate 31. Figure 6 is a dark field photomicrograph illustrating the center of the HRP ejection site in the LH of a representative animal. Ejection site diameter was approximately 500 pm located within the LPAMFB area. Posterior movement of HRP from the site of injection was indicated by extensive labeling of soma and axons located in the dorsal LH-MFB area extending to the posterior MFB. Labeled neurons were also identified in the zona incerta, perifomical, and medial hypothalamic nuclei. Anterior movement was followed along the anterior LHMFB, throughout the LPA-MFB area, amygdaloid nuclei. zona incetta, medial hypothalamic nuclei, stria hypothalamic tract, stria terminalis, nucleus accumbens, and anterior MFB. The most extensive labeling of axons and soma were observed along the LPA-LH-MFB extending to the most anterior portions of the MFB.
Figure 7 illustrates a labeled neuron located in the LPA photographed under light field illumination. The HRP was transported retrogradely to this cell from the ejection site located in the mid LH-MFB area. Many processes in addttion to axons are easily identifiable. Similar neurons were observed in the LH area when HRP was ejected into the LPA-MFB. Figure 8 is a light field photomicrograph of a labeled neuron located in the LH. The HRP ejection site was located in the mid LH-MFB. Neuronal processes are again clearly discemable. Similar neurons were observed in the LPA area when HRP was ejected into the LPA-MFB. Figure 9 illustrates labeled axons located in the MFB photographed under dark field illumination. The HRP was transported retrogradely from the ejection site located in the mid LHMFB. Many individual axons can be identified which in most cases run in parallel. Similar appearing axons were identified both anterior and posterior to ejection sites located in the I.PA and LH regions. I~IS(‘USFION . .
Results indicate intense innervation of both the LPA and by the nucleus accumbens, stria hypothalamic tract. stria terminalis. medial hypothalamic area, zona incerta, and amygdaloid nuclei. These data substantiate the findings of LH
LPA AND LH INTERCONNECTIONS
FIG. 8. Light field photomicrograph of a labeled cell located in the LH. HRP was transported retrogradely to soma from the ejection site located in the mid LH-MFB. Section was processed with DAB for the brown reaction and counterstained with cresyl violet.
FIG. 9. Dark field photomicrograph of labeled axons located in the MFB. HRP ejection site was located in the mid LH-MFB. Section was processed with BDH for the blue reaction and counterstained with neutral red.
187
WAYNER E7’:\1..
IHB
previous studies 18, 12, 13, 14, 15. 18, 19, 21, 28). A large number of cells in either the LPA and LH appear to produce reciprocal innervation extensively throughout both regions. The MFB axons were also stained and densely concentrated throughout both regions. The soma that were observed ag pear to be those of the previously described path neuron [ 131
and located within the MFB. Extensive innervation from other parts of the brain will be described in subsequent publications. In addition, corroborating electrophysiological data and the effects of microiontophoretically ejected HRP at the site of the active neuron will be forthcoming.
REFERENCES I. Almli, C. R. and C. S. Weiss. Drinking behaviors: Effects of lateral preoptic and lateral hypothalamic destruction. Phy.sio/. Rchov. 13: 527-538, 1974. 2. Anand, B. K. and J. R. Brobeck. Hypothalamic control of food intake in rats and cats. Yule J. Biol. Med. 24: 123-140, 1951. 3. Anand, B. K. and R. V. Pillat. Activity of single neurons in the hypothalamic feeding centers: Effects of gastric distension. J.
Bruin Rcs. 67: 21S-238,
Physiol. 192: 63-77, 1967. 4. Barone. F. C., M. J. Wayner.
C. S. Weiss and C. R. Almli. Effects of intragastric water infusion and gastric distension on hypothalamic neuronal activity. Bruin Res. Bull. 4: 267-282. 1979. 5. Barone. F. C., M. J. Wayner, H. U. Aguilar-Baturoni and R. Guevara-Aguilar. Effects of cervical vagus nerve stimulation on hypothalamic neuronal activity. Bruin Rrs. Bull. 4: 381-391. 1979. 6. Blank, D. L. and M. J. Wayner. Lateral preoptic single unit activity: Effects of various solutions. Physiol. Behut,. 15: 72X730, 1975. 7. Blass. E. M. and A. N. Epstein. A lateral preoptic osmosensitive zone for thirst in the rat. J. c.r,rnp. physic;/. P.ryc /rd. 76:
371394. 1971. 8. Dreifuss, J. J., J. T. Murphy and P. Gloor. Contrasting effects of two identified amygdaloid effetent pathways on single hypothalamic neurons. J. Neurophysiol. 31: 237-248, 1968. 9. Epstein, A. N. and P. Teitelbaum. Severe and persistent deficits in thirst produced by lateral hypothalamic damage. In: Thirsr in the Regwlution ofBody Watw. edited by M. J. Wayner. Oxford: Pergamon Press, 1964, pp. 39.tilO. IO. Graybiel, A. M. and M. Devor. A microelectrophoretic delivery technique for use with horseradish peroxidase. Hrtri~r Rc\. 68: 167-173. 1974. I I. Konig, J. R. F. and R. A. Klippel. Thr Rmf Bruin: A .srcv~w~cr.ric~ Atlus
of /hc,
Forebrain
and
Louw
Purrs
r!f‘ thy Bruin
Srcm.
Baltimore: Williams and Wilkins. 1%3. 12. Lammers, H. J. and A. H. M. Lohman. Structure and fiber connections of the hypothalamus of mammals. In: Progrc~.s.t in Bruin
Hewarch.
Vol.
41,
Integrufiw
15. Morgane. P. J. The function of the limbic and rhinic forebrainlimbic midbrain systems and reticular formation in the reguhlion of food and water intake. Ann S. Y. Acntl. .Sci 157: 806848, 1%9. 16. Nauta, W. J. H., M. B. Pritz and R. J. Lasek. Afferents IO the rat caudoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatomical research method.
Hypofhulamic~
Acriviry.
edited by D. F. Swaab and J. P. Schadi. Amsterdam: Elsevier Scientific, 1974, pp. 61-78. 13. Milhouse, 0. E. A Go& study of the descending medial forebrain bundle. Bruin Res. 15: 341-363. 1%9. 14. Morgane, P. J. Limbic-hypothalamic-midbrain interactton in thirst and thirst motivated behavior. In: Thirst in rhr Regularion of Body Wuwr. edited by M. J. Wayner. Oxford: Pergamon Press. 1964, pp. 42e455.
1974.
17. Nicolaidis. S. Discriminatory response of hypothalamic OSmosensitive units to gustatory stimulation in cats. In: Olfilcrio,r rood Tusrr. edited by C. Pfaffmann. New York: Rockefeller University Press, 1969, pp. 569-573. 18. Oomura. Y.. H. Ooyama. 1‘. Yamamoto, t.. Naka. N. Kobayashi and T. Ono. Reciprocal relationship of the lateral and ventromedial hypothalamus in the regulation of food intake. Physic>/. Beha\,.
2: 97-l
IS. 1967.
19 Oomura, Y.. T. Ono and H. Ooyama. Inhibitory action of the amygdala on the lateral hypothalamic area in rats. A’trrrrrr 228: 110&1110. 1970. 20 Oomura, Y., 1‘. Ono. H. Ooyama and M. J. Wayner. Glucose and osmosensitive neurons in the rat hypothalamus. Nrrrrtrc 222: 282-284, l%9. ?I Raisman. G. Neural connections ofthe hypothalamus. Hr. ,&I/. Bull. 22: 197-201, l%6. 22 Teitelbaum, P. and A. N. Epstcm. The lateral hypothalamic syndrome: Recovery of feeding and drinking after lateral hypothalamic lesions. Ps.who/. Ret 69: 74-90, 1962. 23 Tondat. L. M. and C. R. Almli. Fvidence for independent osmosensitivity of the lateral preoptic and lateral hypothalamic neurons. Bruin Rcs. Bull. I: 241-749, 1976. 24 Wayner, M. J. Lateral preoptic-lateral hypothalamic-brain stem motor control system and adjunctive behavior. In: iVrrrrct/ I,rrc,,grurion ofPh.vsiological Mechanism.t und Behavior. edited by G. J. Mogenson and F. R. Calaresu. Toronto: University of Toronto Press. 1975, pp. 396-41 I. 25. Wayner, M. J. Specificity of behavioral regulation. ~/I\..\&. &Izur. 12: 851-869, 1974. 26. Wayner, M. J.. C. C. Loullis and F. C. Barone. Effects of lateral hypothalamic lesions on schedule dependent and schedule induced behavior. Physiol. Behuv. IH: SOF511. 1977. 27. Weiss, C. S. and C. R. Almli. Lateral preoptic and lateral hypothalamic units: In search of the osmoreceptors for thirst. Physiol.
Behut,.
15: 711722.
1975.
2x. Wolf, G. and J. Sutin. Fiber degenerdtion after LH lessons in the rat. .1. c’c,mp. Nerrrol. 127: 137-16. 1966.
