Opposite regulation of body temperature by cholinergic input to the paraventricular nucleus and supraoptic nucleus in rats

Hypothalamic cholinergic system plays an important role in the regulation of body temperature and fluid balance. We have previously shown that cholinergic stimulation of the anterior hypothalamus and preoptic area was accompanied by a fall in body temperature, increased water intake, and increased Fos protein in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). In the present study, to estimate the role played by cholinergic input to the PVN and SON in thermoregulation and water intake, we used microdialysis for cholinergic stimulation with neostigmine and analysis of the nucleus, and also investigated immunoreactivity for c-Fos protein in the brain. This stimulation increased extracellular concentration of acetylcholine in these nuclei. Stimulation of the PVN decreased body temperature and increased water intake. On the other hand, stimulation of the SON increased body temperature. Both in PVN-stimulated and SON-stimulated rats, c-Fos-like immunoreactivity (Fos-IR) was evident in the PVN, SON and certain regions including locus coeruleus (LC), area postrema and nucleus of the solitary tract (NTS). Addition of atropine to the dialysis medium attenuated the increase of Fos-IR and suppressed the cholinergic stimulation-induced responses in body temperature and water intake. These results suggest that cholinergic muscarinic mechanisms in PVN and SON play an opposite function in the regulation of body temperature. The same neuronal pathway including LC and NTS may participate in an advance both in hypothermia and in hyperthermia.     

Effect of lesions of forebrain circumventricular organs on c-fos expression in the central nervous system to plasma hypernatremia

Experiments were carried out on conscious adult male Wistar rats to investigate the effect of selective ablation of the subfornical organ (SFO), and/or the anteroventral third ventricular (AV3V) region on the induction of Fos in central structures in response to plasma hypernatremia. Fos induction, detected immunohistochemically, was used as a marker for neuronal activation. Intravenous infusions of hypertonic saline resulted in dense Fos-like immunoreactivity in several forebrain (paraventricular nucleus of the hypothalamus (PVH), supraoptic nucleus (SON), median preoptic nucleus (MnPO), medial preoptic nucleus, organum vasculosum of the laminae terminalis and SFO) and brainstem (nucleus of the solitary tract, ventrolateral medulla, and parabrachial nucleus) structures. Intravenous infusions of the hypertonic saline solution into animals with lesions of either the SFO, the AV3V or both resulted in a decreased number of Fos-like immunoreactive neurons in the MnPO, PVH and SON. In addition, the number of Fos-labeled neurons in the SON after lesions of both the SFO and the AV3V was significantly greater than that observed in isotonic saline infused controls. Finally, lesions of the forebrain circumventricular structures did not alter the Fos labeling in brainstem structures as a result of the infusion of the hypertonic solution. These data suggest that changes in plasma osmolality and/or concentration of sodium alter the activity of SON and brainstem neurons in the absence of afferent inputs from the SFO and AV3V.     

Distribution of preproadrenomedullin mRNA in the rat central nervous system and its modulation by physiological stressors

Adrenomedullin (ADM), encoded by the preproadrenomedullin (ppADM) gene, exerts multiple effects in a wide variety of peripheral and central tissues. Although ADM-like immunoreactivity has been shown to be widely distributed throughout the rat central nervous system (CNS), the detailed distribution of ppADM gene expression in the CNS and its modulation by physiological stimuli remain unknown. In our study, in situ hybridization was used to localize ppADM mRNA in the rat brain and to quantify its levels after exposure to different stressors including lipopolysaccharide (LPS; 100 microg/kg, iv), restraint stress (2 cycles of 1 hour restraint/1 hour rest), and 24 hours of dehydration. In addition, Fos immunoreactivity was used to identify the activation of neurons in response to LPS. Our results show that ppADM mRNA is widely distributed throughout the rat CNS, with especially high levels in autonomic centers including the hypothalamic paraventricular nucleus (PVN), hypothalamic supraoptic nucleus (SON), locus coeruleus, ventrolateral medulla, and intermediolateral cell column of the spinal cord. Furthermore, LPS inhibits ppADM gene expression in the parvocellular PVN (pPVN), magnocellular PVN (mPVN), SON, dorsal motor nucleus of the vagus, and area postrema among examined regions; restraint stress reduces ppADM mRNA levels in the pPVN, mPVN, SON, nucleus of the solitary tract, dorsal motor nucleus of the vagus, area postrema, and subfornical organ; 24 hours of water deprivation decreases ppADM gene expression only in the mPVN and SON. Taken together, our results suggest that ADM is involved in the regulation of the hypothalamo-neurohypophysial system, the hypothalamo-pituitary-adrenal axis, and central autonomic functions.     

Differences in Fos expression in the rat brains between cold and warm ambient exposures

Fos expression in the rat diencephalon, brain stem, cerebellum, and spinal cord was examined after warm (33°C) and cold (10°C) ambient exposures. Fos expression was examined with use of immunohistochemical method and the number of Fos-positive neurons In each nucleus was quantitatively analyzed. When rats were exposed to cold ambient, significant number of Fos-positive neurons was found in the lateral septal nucleus (LS), preoptic hypothalamic area (POA), parvocallular paraventricular hypothalamic nucleus (PPVN), lateral preoptic area (1-130), zone incerta (ZI), pareventricular thalamic nucleus (PV), ventromedial hypothalamic nucleus (VMH), subparafesclcutm thalamic nucleus (SPF), postrior hypothalamic area (PH), supramammillary nucleus (SuM), microcellular tegmental nucleus (MiTg), lateral iemomscus nucleus (LL), lateral dorsal central grey (CGLD), lateral ventral central grey (CGLV), dorsal parabrachial nucleus (DPS), locus coeruleus (LC), dorsal tegmental nucleus (DTg), vestibular nucleus (Ves), nucleus of solitary tract (Sol), spinal cord, and cerebellum. When animals were exposed to warm ambient, the numbers of Fos-positive neurons in the LS, POA, PV, LPO, and SuM were significantly increased to be equal to those of cold ambient However, after warm ambient exposure the numbers of Fos-positive neurons in the DPB and spinal cord were increased but less than those of cold ambient, and those in the pPVN, VMH, ZI, SPF, PH, CGLD, CGLV, Mg, LL, LC, DTg, Ves, Sol, and cerebellum were not significantly increased as compared with those of control or cold ambient Abdominal temperature was not changed doing cold ambient exposure, but the temperature was sWNcantly increased during warn ambient exposure. These results demonstrate that Fos-positive regions in the rat brains are sagraficantly different between cold and worm ambient exposures although several hypothalamic brain regions reveal the same degree of Fos expression, irxiCating the differences of neural patiwways concerning thennoregulation between cold and warm ambients.     

Effects of Cholecystokinin in the Supraoptic Nucleus and Paraventricular Nucleus are Negatively Modulated by Leptin in 24‐h Fasted Lean Male Rats

Cholecystokinin (CCK) and leptin are two important satiety factors that are considered to act in synergy to reduce meal size. Peripheral injection of CCK activates neurones in several hypothalamic nuclei, including the supraoptic (SON) and paraventricular (PVN) nuclei and neurones in the brainstem of fed rats. We investigated whether peripheral leptin would modulate the effects of CCK on neuronal activity in the hypothalamus and brainstem of fasted rats by investigating Fos expression in the PVN, SON, arcuate nucleus, ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), area postrema (AP) and the nucleus tractus solitarii (NTS). Male rats, fasted for 24 h, received either one i.p. injection of vehicle, leptin or CCK‐8 alone, or received one injection of vehicle or leptin before an i.p. injection of CCK‐8. We found that CCK increased Fos expression in the PVN and SON as well as in the NTS and AP, but had no effect on Fos expression in the arcuate nucleus, VMH or DMH compared to vehicle. Leptin injected alone significantly increased Fos expression in the arcuate nucleus but had no effect on Fos expression in the VMH, DMH, SON, PVN, AP or NTS compared to vehicle. Fos expression was significantly increased in the AP in rats injected with both leptin and CCK compared to rats injected with vehicle and CCK. Unexpectedly, there was significantly less Fos expression in the PVN and SON of fasted rats injected with leptin and CCK than in rats injected with vehicle and CCK, suggesting that leptin attenuated CCK‐induced Fos expression in the SON and PVN. However, Fos expression in the NTS was similar in fasted rats injected with vehicle and CCK or with leptin and CCK. Taken together, these results suggest that leptin dampens the effects of CCK on Fos expression in the SON and PVN, independently from NTS pathways, and this may reflect a direct action on magnocellular neurones.     

Role of preoptic and anterior hypothalamic cholinergic input on water intake and body temperature

To elucidate the role played by cholinergic mechanism in the preoptic area (POA) and anterior hypothalamus (AH) in the control of body temperature and water intake of rats, we used microdialysis without disturbing the behavior of unanesthetized animals. After microdialysis, we also investigated immunoreactivity for c-Fos protein in the hypothalamus. Stimulation with neostigmine, an acetylcholine esterase inhibitor, through microdialysis probe increased extracellular concentration of acetylcholine (ACh) in the POA and AH, and was accompanied by a dose-dependent fall in body temperature and increased water intake. Addition of atropine, a muscarinic receptor antagonist, to the dialysis medium containing neostigmine suppressed the neostigmine-induced changes in rectal temperature and water intake. Neostignime markedly increased c-Fos-like immunoreactivity (Fos-IR) in certain hypothalamic areas, including the paraventricular nucleus, supraoptic nucleus and median preoptic nucleus. This increase was also attenuated by atropine. These results suggest that cholinergic inputs and activation of muscarinic processes in POA and AH induced a decline in body temperature and increased water intake.     

Direct pyrogenic input from prostaglandin EP3 receptor-expressing preoptic neurons to the dorsomedial hypothalamus

Fever is induced by a neuronal mechanism in the brain. Prostaglandin (PG) E2 acts as a pyrogenic mediator in the preoptic area (POA) probably through the EP3 subtype of PGE receptor expressed on GABAergic neurons, and this PGE2 action triggers neuronal pathways for sympathetic thermogenesis in peripheral effector organs including brown adipose tissue (BAT). To explore pyrogenic efferent pathways from the POA, we determined projection targets of EP3 receptor-expressing POA neurons with a special focus on rat hypothalamic regions including the dorsomedial hypothalamic nucleus (DMH), which is known as a center for autonomic responses to stress. Among injections of cholera toxin b-subunit (CTb), a retrograde tracer, into hypothalamic regions at the rostrocaudal level of the DMH, injections into the DMH, lateral hypothalamic area (LH) and dorsal hypothalamic area (DH) resulted in EP3 receptor immunolabelling in substantial populations of CTb-labeled neurons in the POA. Bilateral microinjections of muscimol, a GABA(A) receptor agonist, into the DMH and a ventral region of the DH, but not those into the LH, inhibited thermogenic (BAT sympathetic nerve activity, BAT temperature, core body temperature and expired CO2) and cardiovascular (arterial pressure and heart rate) responses to an intra-POA PGE2 microinjection. Further immunohistochemical observations revealed a close association of POA-derived GABAergic axon swellings with DMH neurons projecting to the medullary raphe regions where sympathetic premotor neurons for febrile and thermoregulatory responses are localized. These results suggest that a direct projection of EP3 receptor-expressing POA neurons to the DMH/DH region mediates febrile responses via a GABAergic mechanism.     

大鼠导水管周围灰质注射谷氨酸可使Barrington核神经元表达Fos

形态学研究已发现大鼠内侧视前区（ＭＰＯ）和导水管周围灰质（ＰＡＧ）发出大量轴突,投射至与排尿反射密切相关的Ｂａｒｒｉｎｇｔｏｎ 核。本研究试图通过注射谷氨酸钠到ＭＰＯ或ＰＡＧ后,观察Ｂａｒｒｉｎｇｔｏｎ 核内的Ｆｏｓ表达情况,来了解以上两通路的性质。将谷氨酸钠注射到ＭＰＯ后,只有少量Ｆｏｓ阳性神经元出现在Ｂａｒｒｉｎｇ－ｔｏｎ 核。而将谷氨酸钠注射到ＰＡＧ后,Ｂａｒｒｉｎｇｔｏｎ 核内出现大量的Ｆｏｓ阳性神经元。此结果提示,ＰＡＧ可能对大鼠脑桥排尿反射活动具有兴奋性调节作用。     

Comparison of c-fos expression in the lamina terminalis of conscious rats after intravenous or intracerebroventricular angiotensin

Fos immunoreactivity in the rat brain after intracerebroventricular (ICV) angiotensin II (ANG II) was compared with that induced by intravenous ANG II. ANG II was infused into the lateral ventricle (at 1 ng/min) or femoral vein (at 5 μg/h) of conscious rats. After 90 min, rats were killed and Fos was detected by immunohistochemistry. Both infusions caused Fos immunoreactivity to be present in the lamina terminalis, hypothalamic supraoptic, and paraventricular nuclei, bed nucleus of the stria terminalis, and central amygdalold nucleus. However, distributions of Fos immunoreactivity within the lamina tenninalis differed with the different routes of infusion. Intravenous ANG II caused intense Fos immunoreactivity mainly in the subfomical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT). By contrast, ICV ANG 11 caused intense Fos immunoreactivity predominantly in the median preoptic nucleus and juxtaventricular neurons of the SFO and OVLT. These results suggest that IV ANG II induces behavioural and endocrine responses by direct actions on the SFO and OVLT, whereas ICV ANG II directly stimulates neurons in the median preoptic nucleus as well neurons in the SFO and OVLT.     

Neuronal expression of fos protein in the forebrain of diabetic rats

We sought to identify the areas that have altered neuronal activity within the hypothalamus of diabetic rats by mapping neuronal expression of c-fos protein (Fos) and Fos-related antigens. After a standard PAP immunocytochemical protocol, Fos-like immunoreactivity was observed in the paraventricular nucleus (PVN), supraoptic nucleus (SON), median preoptic area (MnPO), anterior hypothalamus (AH) and posterior hypothalamus (PH) of control (vehicle; n=6) and diabetic rats (Sprague-Dawley rats injected with STZ 65 mg/kg/ip 4 weeks prior to the experiment; n=6). Blood glucose levels were significantly elevated in the diabetic group (370+/-8 mg/dl) compared to control group (104+/-3 mg/dl). Diabetic rats had a significantly higher number of Fos-positive cells in PVN (2.5x), SON (7x) and MnPO (2x) compared to the control rats. However, diabetic rats had significantly fewer Fos-positive cells in the AH (0.3x) and no difference was observed in the PH between the diabetic and control rats. Despite the elevated number of Fos-positive cells in the diabetic rats, dehydration (water withdrawal for 24 h) or hypertonic challenge (1.5 ml of 0.1 M NaCl i.p. injection) produced a further increase in the number of Fos-positive cells in the PVN, SON and MnPO. Dehydration did not alter the number of Fos-positive cells in the AH or PH, but hypertonic challenge produced a significant increase in the Fos-positive cells in both the AH and PH of diabetic rats. This study demonstrates that: (1) there is increased basal neuronal activity in the PVN, SON and MnPO, a decrease in neuronal activity in the AH and no change in neuronal activity in the PH as indicated by Fos staining in diabetic rats; and (2) dehydration or hypertonic challenge produces a further increase in the number of Fos-positive cells in the PVN, SON, and MnPO which is comparable to control rats. These data support the conclusion that vasopressin producing neurons in the PVN and SON and autonomic areas within the lamina terminalis and hypothalamus are activated during diabetes and may contribute to the elevated levels of vasopressin and autonomic dysfunction during diabetes.     

Evidence for reciprocal connections between the dorsochiasmatic area and the hypothalamo neurohypophyseal system and some related extrahypothalamic structures

In the preceeding article, a dorsochiasmatic area (DCh) was described that projects to both paraventricular (PVN) and supraoptic (SON) nuclei. The main afferents of the DCh, revealed by local injections of retrograde tracers, are the hypothalamic PVN and SON, lateral septal nuclei (LSV and SHy), bed nuclei of the stria terminalis (BST), anteroventral third ventricle region, particularly the median preoptic nucleus (MnPO), the subfornical organ, medial preoptic areas, arcuate hypothalamic nucleus, ventromedial hypothalamic nuclei, paraventricular thalamic nucleus, and, more caudally, several structures of the posterior hypothalamus and mesencephalon. The relations between DCh and BST, LSV, SHy, or MnPO appeared reciprocal. In view of their reciprocal relationships with the hypothalamo-neurohypophyseal system and some of their related extrahypothalamic structures, the DCh might be involved in the regulation of the vasopressin (AVP) and/or oxytocin (OT) systems, or in reproductive behavior.     

Fos expression following self-stimulation of the medial prefrontal cortex

Self-stimulation of the medial prefrontal cortex and medial forebrain bundle appears to be mediated by different directly activated fibers. However, reward signals from the medial prefrontal cortex do summate with signals from the medial forebrain bundle, suggesting some overlap in the underlying neural circuitry. We have previously used Fos immunohistochemistry to visualize neurons activated by rewarding stimulation of the medial forebrain bundle. In this study, we assessed Fos immunolabeling after self-stimulation of the medial prefrontal cortex. Among the structures showing a greater density of labeled neurons in the stimulated hemisphere were the prelimbic and cingulate cortex, nucleus accumbens, lateral preoptic area, substantia innominata, lateral hypothalamus, anterior ventral tegmental area, and pontine nuclei. Surprisingly, little or no labeling was seen in the mediodorsal thalamic nucleus or the locus coeruleus. Double immunohistochemistry for tyrosine hydroxylase and Fos showed that within the ventral tegmental area, a substantial proportion of dopaminergic neurons did not express Fos. Despite previous suggestions to the contrary, comparison of the present findings with those of our previous Fos studies reveals a number of structures activated by rewarding stimulation of both the medial prefrontal cortex and the medial forebrain bundle. Some subset of activated cells in the common regions showing Fos-like immunoreactivity may contribute to the rewarding effect produced by stimulating either site.     

Functional and anatomic relationship between cholinergic neurons in the median preoptic nucleus and the supraoptic cells

The median preoptic nucleus (MePO) has been suggested to be an important area in the brain for the regulation of vasopressin (VP) release under the condition of osmotic stimulation. Fos immunoreactivity (Fos-ir), choline acetyltransferase (ChAT) immunoreactivity and retrograde labeling with fluoro-gold were used in this study to determine whether cholinergic neurons in the MePO can be activated by hypertonic NaCl, and to characterize the specific MePO cells that have anatomic projections to the supraoptic nuclei (SON). The results showed that c-fos expression specifically induced by hypertonic NaCl was found in the ChAT cells of the MePO. A retrograde tracing experiment demonstrated that the MePO neurons projecting to the SON were cholinergic. In addition, hypertonic saline-induced Fos-ir was colocalized with the MePO neurons back labeled with fluoro-gold from the SON. Together, these data provide evidence that the MePO cholinergic neurons are activated by osmotic stimulation, and suggest that cholinergic cells in the MePO are functionally important in the control of the SON neurons under the condition of hypertonic stimulation.     

Afferent connections of the rat''s supraoptic nucleus

Neurons projecting to the supraoptic nucleus (SON) have been identified following stereotaxic injections of either horseradish peroxidase or fast blue into the SON region of adult rats. The subfornical organ, median preoptic nucleus, organum vasculosum of the lamina terminalis and medial septal nucleus were the source of the largest numbers of supraoptic-projecting neurons. Several smaller projections also originate from the ipsilateral locus coeruleus, preoptic area, lateral parolfactorial area, dorsomedial nucleus of the hypothalamus, lateral parabrachial nucleus and ventrolateral medulla. Several other areas appeared to project only to the region immediately dorsal to the SON: lateral septal nucleus, diagonal band of Broca, ventral tegmental nucleus, and the supramamillary nucleus. These areas may influence SON neurosecretory function by way of interneurons found immediately dorsal to SON. Additional areas were identified with retrograde fluorescent label only, and these projected to the area immediately dorsal to SON and/or to SON itself.     

A functional neuroanatomical investigation of the role of the medial preoptic area in neural circuits regulating maternal behavior

The medial preoptic area (MPOA) is essential for normal maternal behavior in the rat. Hormone stimulation of the MPOA facilitates the behavior and lesions of the MPOA and the adjoining ventral part of the bed nucleus of the stria terminalis (vBST) disrupt the behavior. The MPOA/vBST also show increases in Fos protein expression during maternal behavior. The present study examines the larger neural circuitry within which the MPOA/vBST might operate to influence maternal behavior. Combining Fos immunocytochemistry with unilateral excitotoxic amino acid lesions or lateral knife cuts of the MPOA/vBST, we sought to identify brain regions which might be under the influence of Fos expressing neurons in the MPOA/vBST. Two brain regions, the shell of the nucleus accumbens (NAs), and the intermediate part of the lateral septum (LSi) were identified. Both the NAs and LSi exhibited elevated Fos expression during maternal behavior, while unilateral MPOA/vBST damage resulted in an ipsilateral reduction of maternal behavior-induced Fos expression in each area, suggesting that MPOA/vBST neurons modulate Fos expression and associated neural activity in both of these structures during maternal behavior. Importantly, these unilateral preoptic lesions also depressed maternal behavior-induced Fos expression in the ipsilateral MPOA and vBST. The effects of these lesions on Fos expression in the periaqueductal gray (PAG) and other brain regions are also presented.     

Fos expression in rat pontine tegmental neurons following activation of the medial preoptic area

Fos immunohistochemistry was used to map the distribution of pontine neurons excited by activation of the medial preoptic area (MPO). Although we have previously shown that Barrington's nucleus receives a very dense focal input from the MPO, electrical stimulation of the preoptic area unexpectedly induced very little Fos expression in Barrington's neurons. These results suggest that the MPO→Barrington's projection utilizes a transmitter(s) that does not involve transduction of the Fos protein; alternatively, MPO afferents to Barrington's nucleus may be inhibitory in nature. As Barrington's nucleus plays a critical role in micturition, MPO projections to Barrington's nucleus may regulate voiding reflexes during sexual behavior. Interestingly, while the locus coeruleus (LC) proper receives only a sparse projection from the MPO, extensive Fos expression was present in LC. The finding of Fos immunoreactive LC neurons suggests that the excitatory influence of MPO may regulate LC neuronal activity and NE release during reproductive behaviors.     

Projections of the posterodorsal preoptic nucleus and the lateral part of the posterodorsal medial amygdala in male gerbils, with emphasis on cells activated with ejaculation

The posterodorsal preoptic nucleus (PdPN) and the lateral part of the posterodorsal medial amygdala (MeApd) express Fos with ejaculation in male gerbils. Ejaculation-activated cells participate in the PdPN and MeApd projections to each other and to the sexually dimorphic preoptic area (SDA), but those projections involve less than 20% of the activated PdPN cells and less than 50% of the activated MeApd cells. To identify other potential targets of ejaculation-activated cells, we traced PdPN and lateral MeApd outputs using biotinylated dextran amine. The principal part of the bed nucleus of the stria terminalis (BSTpr) and the anteroventral periventricular nucleus (AVPv) were labeled from both sites and were injected with Fluoro-Gold to determine whether PdPN and lateral MeApd cells that express Fos with ejaculation would be retrogradely labeled. Fluoro-Gold was also applied to the dorsomedial hypothalamus (DMH) and retrorubral field (RRF) because such injections label PdPN cells in rats. The PdPN-DMH projection is minimal in gerbils, involving few, if any, ejaculation-related cells. Ejaculation-activated PdPN cells project to the AVPv (43%), dorsal BSTpr (30%), and RRF (12%). Those in the lateral MeApd project to the dorsal BSTpr (43%) and AVPv (18%). When these percentages are combined with those for ejaculation-activated cells involved in the PdPN and lateral MeApd projections to each other and to the medial SDA, the totals reach 100%. Thus, every PdPN and MeApd cell activated with ejaculation may participate in one of these projections. Similar projections may contribute to the similar behavioral effects of the PdPN and MeApd.     

Reciprocal connections between the medial preoptic area and the midbrain periaqueductal gray in rat: a WGA-HRP and PHA-L study.

The midbrain periaqueductal gray (PAG) participates in diverse functions such as analgesia, autonomic regulation, sexual behavior, and defense/escape responses. Anatomical studies of the circuits involved in such functions have largely focused on the connections of PAG with the medulla. Projections to PAG from forebrain structures are extensive, but their organization has received little attention. Previous anatomic studies indicate that the medial preoptic area (MPO), involved in a variety of physiological and behavioral functions, is a major source of afferent input to the periaqueductal gray. Here, we have examined the topography of reciprocal connections between these two structures in the rat by using wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) and Phaseolus vulgaris leucoagglutinin (PHA-L). Multiple WGA-HRP injections at several rostrocaudal levels of PAG retrogradely labeled large numbers of neurons in the medial preoptic area; labeled cells were primarily located in the medial preoptic nucleus, the median preoptic nucleus, and the region lateral to the medial preoptic nucleus. The distribution of labeled cells shifted medially to laterally along the rostral to caudal axis of the medial preoptic area. Rostrally, there was selective retrograde labeling in the central and lateral divisions of medial preoptic nucleus, whereas caudally, labeled cells were primarily located only in the lateral subdivision of medial preoptic nucleus. Tracer injections in PAG also produced strong anterograde labeling in MPO. WGA-HRP and PHA-L injections in the medial preoptic area resulted in dense anterograde labeling along the entire rostrocaudal axis of PAG. The terminal labeling in PAG from the medial preoptic area was not uniformly distributed throughout PAG, however. Instead, this projection formed one or two rostrocaudally oriented longitudinal columns that terminated in different subregions of PAG along the entire rostrocaudal axis of this structure. Rostrally, inputs from the medial preoptic area project heavily to dorsomedial PAG, and at mid-PAG levels, the projection becomes distinctly bipartite with two discrete longitudinal terminal columns in dorsomedial and lateral PAG; caudally, the heaviest labeling is in ventrolateral PAG. The projection also exhibited a central to peripheral (radial) gradient; labelled fibers and terminals were heaviest near the aqueduct and much lower in the peripheral parts of PAG. WGA-HRP injections in MPO also produced retrograde labeling of neurons at all rostrocaudal levels of PAG; more neurons were labeled in the rostral than the caudal half of PAG. The majority of labeled cells were located in dorsomedial and ventral/ventrolateral parts of PAG; only a few neurons in the dorsal raphe region appear to project to MPO.(ABSTRACT TRUNCATED AT 400 WORDS)