Anatomical distribution and biochemical characterization of the novel RFamide peptide 26RFa in the human hypothalamus and spinal cord.

26RFa is a novel RFamide peptide originally isolated in the amphibian brain. The 26RFa precursor has been subsequently characterized in various mammalian species but, until now, the anatomical distribution and the molecular forms of 26RFa produced in the CNS of mammals, in particular in human, are unknown. In the present study, we have investigated the localization and the biochemical characteristics of 26RFa-like immunoreactivity (LI) in two regions of the human CNS--the hypothalamus and the spinal cord. Immunohistochemical labeling using specific antibodies against human 26RFa and in situ hybridization histochemistry revealed that in the human hypothalamus 26RFa-expressing neurons are located in the paraventricular and ventromedial nuclei. In the spinal cord, 26RFa-expressing neurons were observed in the dorsal and lateral horns. Characterization of 26RFa-related peptides showed that two distinct molecular forms of 26RFa are present in the human hypothalamus and spinal cord, i.e. 26RFa and an N-terminally elongated form of 43 amino acids designated 43RFa. These data provide the first evidence that 26RFa and 43RFa are actually produced in the human CNS. The distribution of 26RF-LI suggests that 26RFa and/or 43RFa may modulate feeding, sexual behavior and transmission of nociceptive stimuli.

Cyto- and chemoarchitecture of the hypothalamus of a wallaby ( Macropus eugenii) with special emphasis on oxytocin and vasopressinergic neurons.

We have studied the organization of the hypothalamus in an Australian diprotodontid metatherian mammal, the wallaby ( Macropus eugenii), using cytoarchitectural, histochemical and immunohistochemical techniques. Coronal sections of adult brains were processed for Nissl staining, histochemical reactivity (cytochrome oxidase, nicotinamide adenine dinucleotide phosphate diaphorase and acetylcholinesterase) and immunohistochemistry (antibodies to tyrosine hydroxylase, calbindin, calretinin, non-phosphorylated neurofilament protein, oxytocin and vasopressin). The distribution of immunoreactive neurons for these substances was mapped with the aid of a computer-linked microscope. In general, the wallaby hypothalamus showed a similar nuclear organization to that seen in rodents. The paraventricular nucleus could be divided into several subdivisions based on the different cellular parcellation, similar to that described in rodents. The ventromedial hypothalamic nucleus had cell-sparse dorsomedial and cell-dense ventrolateral subdivisions as seen in eutheria, suggesting a similar functional compartmentalization in all theria. The positions of tyrosine hydroxylase-positive neurons in the wallaby hypothalamus were also similar to those in eutheria. Oxytocin and vasopressinergic neurons were found in all the same major nuclear groups as seen in eutheria, although a nucleus circularis could not be identified. The general similarities between wallaby and eutherian hypothalamus indicate that the basic chemo- and cytoarchitectural features of the hypothalamus are common to eutheria and metatheria and validate the use of the wallaby as a mammalian model of wide applicability in investigations of hypothalamic functional development.

Hypothalamopontine projections in the rat: anterograde axonal transport studies utilizing light and electron microscopy.

Projections to the basilar pontine nuclei (BPN) from a variety of hypothalamic nuclei were traced in the rat utilizing the anterograde transport of biotinylated dextran amine. Light microscopy revealed that the lateral hypothalamic area (LH), the posterior hypothalamic area (PH), and the medial and lateral mammillary nuclei (MMN and LMN) are the four major hypothalamic nuclei that give rise to labeled fibers and terminals reaching the rostral medial and dorsomedial BPN subdivisions. Hypothalamopontine fibers extended caudally through the pontine tegmentum dorsal to the nucleus reticularis tegmenti pontis and then coursed ventrally from the main descending bundle toward the ipsilateral basilar pontine gray. Some hypothalamopontine fibers crossed the midline in the tegmental area just dorsal to the pontine gray to terminate in the contralateral BPN. Electron microscopy revealed that the ultrastructural features of synaptic boutons formed by axons arising in the LH, PH, MMN, and LMN are similar to one another. All labeled hypothalamopontine axon terminals contained round synaptic vesicles and formed asymmetric synaptic junctions with dendritic shafts as well as dendritic appendages, and occasionally with neuronal somata. Some labeled boutons formed the central axon terminal in a glomerular synaptic complex. In summary, the present findings indicate that the hypothalamus projects predominantly to the rostral medial and dorsomedial portions of the BPN which, in turn, provide input to the paraflocculus and vermis of the cerebellum. Since the hypothalamic projection zones in the BPN also receive cerebral cortical input, including limbic-related cortex, the hypothalamopontine system might serve to integrate autonomic or limbic-related functions with movement or somatic motor-related activity. Alternatively, since the cerebellum also receives direct input from the hypothalamus, the BPN may function to provide additional somatic and visceral inputs that are used by the cerebellum to perform the integrative function.

Changes in hypothalamic prostaglandin E2 may predict the occurrence of sleep or wakefulness as assessed by parallel EEG and microdialysis in the rat.

Prostaglandin (PG) E2 is produced by mammalian hypothalamus and when administered exogenously prolongs wakefulness. In order to study the relation of endogenous hypothalamic PGE2 to sleep and wakefulness, we have used microdialysis in freely moving rats associated with EEG recording. Male Wistar rats were implanted with three cortical electrodes and with a guide cannula for microdialysis in the space between the paraventricular nucleus (PVN) and the ventromedial hypothalamus (VMH). PGE2 was measured by RIA in 3- or 6-min dialysates 15 days after surgery, when sleep patterns were normal again and PGE2 production stabilised. PGE2 levels were significantly higher during wakefulness (601 +/- 35 pg/ml, 5 experiments, 35 samples) than during slow-wave sleep (487 +/- 24 pg/ml, 5 experiments, 49 samples). Samples corresponding to paradoxical sleep showed a tendency towards higher PGE2 values compared to slow-wave sleep but lower compared to wakefulness. In epochs of wakefulness or sleep lasting at least 12 min, high PGE2 levels in the middle of wakefulness regularly dropped, thus announcing the occurrence of sleep. During sleep, PGE2 first went on dropping and then reincreased towards the values that characterize early periods of wakefulness. In its turn, this reincrease in PGE2 announced the end of sleep and the imminent occurrence of wakefulness. It is the first study to our knowledge showing that the evolvement in endogenous PG profile may predict the occurrence of sleep or wakefulness.

Cranial and cerebral-ventricular landmarks for accurate stereotaxic approach to hypothalamic nuclei in the goat brain.

This paper describes a new stereotaxic coordinate system for the goat brain based on cranial landmarks. An osseous triangle (a-b-c) formed by the point of junction of the crista galli with the caudal ventral part of the frontal sinus septum (a), the external occipital protuberance (b), and the midsagittal projection of the external acoustic meatus (c), was measured using lateral radiographs and ventriculographs and showed a constant mathematical relation. The rostral angle was 20.3 +/- 1.0 (mean +/- SD) degree in 23 goats studied regardless of their cranial size which varied considerably from one animal to another. The hypotenuse length (a-b distance) was found to be a good predictor of the rostral nuchal position of the anterior commissure (AC) and the infundibular recess of the third ventricle (INF), by which the individual variance of the stereotaxic coordinates for a given hypothalamic structure could be compensated. The anterior-posterior distances from the external acoustic meatus was highly correlated with the a-b distance for AC (r = 0.88) and INF (r = 0.90). Using these cranial landmarks and the method outlined in this paper, uncertainty in coordinate values for AC and INF in the goat brain was reduced considerably in comparison to deviation observed when the ordinary Horsley-Clarke axis (Reid's plane) was employed.

Anorexic action of a new potential neuropeptide Y antagonist [D-Tyr27,36, D-Thr32]-NPY (27-36) infused into the hypothalamus of the rat.

Neuropeptide Y (NPY) produces a vigorous feeding response in several species when it is injected into hypothalamic structures involved in eating behavior. The purpose of this study was to determine whether a unique carboxy terminal fragment of NPY would alter the pattern of eating induced in the rat either by NPY injected into the hypothalamus or by a 24-h period of food deprivation. In this case, two L-tyrosine residues and one L-threonine residue of the NPY27-36 fragment were transformed to their D-conformation to produce [D-Tyr27,36,D-Thr32]-NPY (27-36), i.e., D-NPY27-36. Guide cannulae for microinjection were implanted stereotaxically just dorsal to the paraventricular nucleus (PVN) or ventromedial hypothalamus (VMH) of 24 adult male Sprague-Dawley rats. Following postoperative recovery, a microinjection of artificial CSF or 1.1 microgram or 3.3 micrograms of a peptide was made directly into the PVN or VMH as follows: native NPY; D-NPY27-36; or [L-Tyr27,36, L-Thr32]-NPY (27-36), i.e., L-NPY27-36. Food intakes were measured at intervals of 0.25, 0.5, 1.1, 2.0, 4.0, and 24 h. When D-NPY27-36 was microinjected at NPY reactive sites in the PVN or VMH of the rat 15 min before a similar microinjection of NPY, the intense eating response induced by the peptide was reduced significantly. Not only was the effect dose dependent, but D-NPY27-36 also augmented the latency to feed. A mixture of the two doses of NPY and D-NPY27-36 injected at the same hypothalamic loci did not attenuate the intake of food but tended to enhance the feeding response in the rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex differences in the distribution and projections of testosterone target neurons in the medial preoptic area and the bed nucleus of the stria terminalis of rats.

Retrograde tracing was combined with steroid hormone autoradiography to investigate the projections of testosterone-target neurons in preoptic and limbic regions to the midbrain in male and female rats. Autoradiograms were prepared from the brains of male and female rats that had received an injection of a fluorescent retrograde tracer into the midbrain, and an intravenous injection of [3H]testosterone. Testosterone target neurons that project to the midbrain were abundant in the medial preoptic nucleus (MPN) and bed nucleus of the stria terminalis (BST) and were also observed in the ventromedial nucleus of the hypothalamus. Testosterone target neurons and testosterone target neurons that project to the midbrain were more abundant in the caudal half of the MPN compared to the rostral half. Moreover, male rats had more testosterone target neurons in the caudal MPN than female rats, and the number of testosterone target neurons in the MPN that project to the midbrain was higher in male than in female rats. Male rats also had more testosterone target neurons than females throughout the encapsulated subdivision of the BST. We hypothesize that sex differences in the neuronal connectivity of testosterone target neurons may underly sex specific behavioral responsiveness to androgens.

Efferent connections of the hypothalamic "aggression area" in the rat.

The efferent connections of the hypothalamic area of the rat, where attack behaviour can be elicited by electrical stimulation, were studied using iontophoretic injections of Phaseolus vulgaris-leucoagglutinin. Specificity for the hypothalamic "attack area" was investigated by comparison with efferents of hypothalamic sites outside the attack area. The hypothalamic attack area consists of the intermediate hypothalamic area and the ventrolateral pole of the ventromedial hypothalamic nucleus. Fibres from the hypothalamic attack area, as well as fibres from several other hypothalamic sites, form diffuse fibre "streams" running rostrally or caudally. Many varicosities that are found on the fibres suggest, that these fibres are capable of influencing many brain sites along their way. Projection sites were found throughout the brain. In the comparison between attack area efferents and controls, many overlapping brain sites were found. Hypothalamic efferents preferentially originating in the largest part of the attack area, i.e. the intermediate hypothalamic area, were found in the mediodorsal and parataenial thalamic nuclei. Within the septum, a spatial organization of hypothalamic innervation was found. Fibres from the attack area formed specialized "pericellular baskets" in the dorsolateral aspect of the intermediate part of the lateral septal nucleus. Fibres from other hypothalamic sites were found in other septal areas and did not form these septal baskets. Within the mesencephalic central gray, fibres from the attack area were found specifically in the dorsal part and dorsal aspect of the lateral part of the central gray. Physiological and pharmacological studies have shown that several brain sites are involved in different aspects of aggressive behaviour. Some of these areas, as for instance the dorsomedial thalamic nucleus, septum and central gray, are innervated by efferents from the hypothalamic attack area, whereas other sites, like ventral premammillary nucleus and ventral tegmental area, are not. It is concluded from the present findings, that a number of brain sites, that are known to be involved in agonistic behaviour, receive hypothalamic information preferentially from the hypothalamic attack area through diffusely arranged varicose fibres. The function of each connection in the regulation of specific behaviours remains to be further investigated.

Prostaglandins and hypothalamic neurotransmitter receptors involved in hyperthermia: a critical evaluation.

The role of a prostaglandin of the E series (PGE) in the hypothalamic mechanisms underlying a fever continues to be controversial. This paper reviews the historical literature and current findings on the central action of the PGEs on body temperature (Tb). New experiments were undertaken to examine the local effect of muscarinic, nicotinic, serotonergic, alpha-adrenergic, or beta-adrenergic receptor antagonists at hypothalamic sites where PGE1 caused a rise in Tb of the primate. Guide tubes for microinjection were implanted stereotaxically above sites in and around the anterior hypothalamic, preoptic area (AH/POA) of male Macaque monkeys. Following postoperative recovery, 30-100 ng of PGE1 was micro-injected unilaterally in a volume of 1.0-1.5 microliter at sites in the AH/POA to evoke a rise in Tb, and once identified, pretreated with a receptor antagonist. PGE1 hyperthermia was significantly reduced by microinjections of the muscarinic and nicotinic antagonists, atropine, or mecamylamine, at PGE1 reactive sites in the AH/POA. The serotonergic antagonist, methysergide, injected at PGE1 sensitive sites in the ventromedial hypothalamus also attenuated the rise in Tb. However, the 5-HT reuptake blocker, fluoxetine, and the beta-adrenergic receptor antagonist, propranolol, injected in the AH/POA failed to alter the PGE1 hyperthermia. In contrast, the alpha-adrenergic antagonist, phentolamine, potentiated the increase in Tb at all PGE1 reactive sites in the hypothalamus. An updated model is presented to explain how the concurrent actions of aminergic neurotransmitters acting on their respective receptors in the hypothalamus can interact with a PGE to elicit hyperthermia. Finally, an evaluation of the current literature including recent findings on macrophage inflammatory protein (MIP-1) supports the conclusion that a PGE in the brain is neither an obligatory nor essential factor for the expression of a pyrogen fever.

Hypothalamic involvement in stress-induced hypocalcemia in rats.

Although hormonal regulation of blood calcium homeostasis has been intensively investigated in the peripheral organs, the involvement of the central nervous system in calcium regulation is still poorly understood. In the present study, we found that (1) bilateral lesions of the ventromedial nucleus of the hypothalamus (VMH), but not those of the paraventricular hypothalamic nucleus or the lateral hypothalamic area, eliminated immobilization (IMB)-induced hypocalcemia, and (2) electrical stimulation of the VMH decreased the blood calcium level. The results suggest that the VMH has a hypocalcemic function and plays a role in IMB-induced hypocalcemia.

Identification, partial characterization, and hypothalamic distribution of kappa, mu, and delta opioid receptors in a passerine songbird (Junco hyemalis).

Brain tissues obtained from a passerine songbird (dark-eyed junco, Junco hyemalis) were used to identify and partially characterize central opioid receptors. We found that [3H]EKC (putative kappa ligand), [3H]DAMGO (putative mu ligand), and [3H]DPDPE and [3H]pCl-DPDPE (two putative delta ligands) bind to brain tissue preparations specifically, in a time-dependent fashion, and with a high affinity (Kd's < or = 5 nM). Binding sites are present at low concentrations (Bmax < 120 fmol/mg protein), and they are pharmacologically selective. In vitro autoradiography studies revealed a high density of delta receptors in hypothalamic regions (ventromedial and lateral hypothalamus) that regulate feeding behavior. Together with previous studies, these observations suggest that the central influence of opioids on avian food consumption depends on mechanisms located in hypothalamic regions.

Activation of the satiety center by auricular acupuncture point stimulation.

Stimulation of the rat inner auricular regions that correspond to the human pylorus, lung, trachea, stomach, esophagus, endocrine, and heart acupuncture points evoked potentials in the hypothalamic ventromedial nucleus (HVM), the satiety center. Needle implantation into any of these points reduced the body weight to its initial 290 g after the rat had gained about 410 g in 20 days, and significantly reduced initial 450-g body weights (p less than 0.01, Student's t test) in 14 days. Stimulation of other acupuncture points did not evoke HVM potentials and did not reduce body weight. After the HVM was lesioned, body weight increased and acupuncture point needling had no effect on body weight. Needling of the auricular acupuncture points evoked no potentials in the lateral hypothalamus (LHA), the feeding center, and had almost no influence on weight reduction induced by LHA lesion.

Effects of periodontal stimulation on VHM neurones in anesthetized rats.

In anesthetized rats, mechanical stimulations, the direction and intensity of which could be modified, were applied to the superior incisive while single neurone activities were recorded within the VHM region by means of extracellular glass microelectrodes. The great sensitivity of periodontal receptors to the direction and the intensity of the stimulations was demonstrated at this level. This finding, which is reported here in detail for the first time, suggests that the periodontal afferents might be involved in physiological mechanisms including food intake regulation and osteomorphogenesis.

Neuroanatomical specificity in the co-localization of aromatase and estrogen receptors.

The relative distributions of aromatase and of estrogen receptors were studied in the brain of the Japanese quail by a double-label immunocytochemical technique. Aromatase immunoreactive cells (ARO-ir) were found in the medial preoptic nucleus, in the septal region, and in a large cell cluster extending from the dorso-lateral aspect of the ventromedial nucleus of the hypothalamus to the tuber at the level of the nucleus inferioris hypothalami. Immunoreactive estrogen receptors (ER) were also found in each of these brain areas but their distribution was much broader and included larger parts of the preoptic, septal, and tuberal regions. In the ventromedial and tuberal hypothalamus, the majority of the ARO-ir cells (over 75%) also contained immunoreactive ER. By contrast, very few of the ARO-ir cells were double-labeled in the preoptic area and in the septum. More than 80% of the aromatase-containing cells contained no ER in these regions. This suggests that the estrogens, which are formed centrally by aromatization of testosterone, might not exert their biological effects through binding with the classical nuclear ER. The fact that significant amounts of aromatase activity are found in synaptosomes purified by differential centrifugation and that aromatase immunoreactivity is observed at the electron microscope level in synaptic boutons suggests that aromatase might produce estrogens that act at the synaptic level as neurohormones or neuromodulators.

A three-dimensional reconstruction of the rat hypothalamus.

Tracings of structures present in serial coronal frozen sections of the rat hypothalamus were entered into an IBM-PC and sections were aligned in space using a program for 3-dimensional reconstruction. The positions and relative volumes of 16 major hypothalamic nuclei were accurately displayed in lateral, medial, and superior views of the hypothalamus. Three major clusters of hypothalamic nuclei were apparent, reinforcing embryological concepts of "neuromeres" from which adult structures develop. A better knowledge of the spatial locations of hypothalamic nuclei, which determine the pathways of intrahypothalamic connections, should be of aid in interpreting studies which disrupt such connections.

Sexually dimorphic areas in the brain of whiptail lizards.

The whiptail lizard species Cnemidophorus inornatus exhibits sexually dimorphic mating behaviors. We report that complementary sexual dimorphisms exist in two hypothalamic regions in male and female C. inornatus. The anterior hypothalamus-preoptic area (AH-POA), which is involved in male-typical mounting and intromission behaviors, is larger in males. The ventromedial hypothalamus (VMH), which controls female-typical receptivity, is larger in females. The all-female whiptail lizard Cnemidophorus uniparens, a direct descendant of C. inornatus, regularly and reliably displays both male-like mounting and female-like receptive (pseudosexual) behaviors. Rather than having a male-like AH-POA, however, the AH-POA as well as the VMH of C. uniparens resemble that of female C. inornatus. The biological basis of pseudosexual behavior in the parthenogen appears to be due to an unusual sensitivity to ovarian progesterone rather than to a difference in brain morphology.

Phaseolus vulgaris leuco-agglutinin tracing of intrahypothalamic connections of the lateral, ventromedial, dorsomedial and paraventricular hypothalamic nuclei in the rat.

Intrahypothalamic connections of the lateral (LHA), ventromedial (VMH), dorsomedial (DMH) and paraventricular (PVN) hypothalamic nuclei were studied with anterograde transport of iontophoretically injected Phaseolus vulgaris leuco-agglutinin and the immunocytochemical detection of labeled structures. The LHA was found to give rise to a minor projection in the VMH, whereas the VMH in reverse maintains few connections with the ventromedial part of the tuberal LHA. Tracer deposits in both the LHA and VMH resulted in anterograde terminal labeling in the DMH. The DMH, in turn, donates a small number of projections to the LHA and VMH. The main projection of the DMH is aimed at the parvocellular paraventricular nucleus. Direct outflow pathways from the VMH to the PVN were not found, but lectin injections in the LHA on the other hand gave rise to terminal labeling in both the parvocellular and magnocellular divisions of the PVN. The PVN in turn was found to give only minor reciprocal projections to the LHA, DMH and VMH. These findings indicate that the main stream of connections in the hypothalamus runs from the LHA and VMH to the DMH, and from the DMH to the PVN. The identified circuitry patterns were discussed with respect to the role of the hypothalamus in the control of homeostasis and metabolic regulation, and more specifically in relation to the modulation of the hormone release from the pancreas and adrenal glands.

Topographic organization of projections from the amygdala to the hypothalamus of the rat.

Afferent fibers from the amygdala to subdivisions of lateral, ventromedial and dorsomedial hypothalamic nuclei were investigated in rat by retrograde transport of horseradish peroxidase. Small (intranuclear size) peroxidase deposits were placed in hypothalamic nuclei by iontophoresis of a tracer solution containing poly-L-alpha-ornithine which greatly limited diffusion. The medial, central and amygdalo-hippocampal nuclei of the amygdala were found to be the major donors of amygdaloid afferent fibers to the hypothalamus, but there was also substantial labeling of somata in cortical, basomedial, basolateral and lateral amygdaloid nuclei and the intra-amygdaloid bed nucleus of the stria terminalis. No fibers projected from the posterior cortical nucleus of the amygdala to the hypothalamus. Most amygdaloid projections to the lateral hypothalamic area originated in the anterior half of the amygdala, while projections to the ventromedial hypothalamic nucleus arose along the entire length of the amygdala except the posterior cortical nucleus. The amygdalo-hippocampal area projects to the medial hypothalamus. Other amygdaloid nuclei project to both the medial and lateral hypothalamic nuclei. These topographic organizations of amygdaloid afferent fibers to various subdivisions of the hypothalamic nuclei are discussed and compared with other anatomical studies on these connections.

Descending pathways from hypothalamus to dorsal motor vagus and ambiguus nuclei in the rat.

The anatomical pathways between the hypothalamus and cell groups of the lower medulla that are involved in the neural control of endocrine pancreas activity were investigated. As part of this control system the descending pathways originating from lateral, dorsomedial and ventromedial hypothalamic nuclei towards the dorsal motor vagus and ambiguus nuclei, were studied by retrograde transport of horseradish peroxidase. Very small injections of the tracer, by means of the iontophoretic delivery method, were placed in the dorsal motor vagus, ambiguus and solitary tract nucleus as well as in the various nuclei of the medullary reticular formation. Subsequent retrograde labeling was studied in the hypothalamus and the brainstem. The appearance of considerable retrograde labeling in mesencephalic periventricular grey and rostral mesencephalic reticular formation indicated a possible role for these structures as intermediates in an indirect hypothalamo-medullary control circuitry. This led us to extend the peroxidase injections to these mesencephalic areas after which the hypothalamus was investigated for retrograde labeling. All data combined indicated the existence of three descending pathways, direct and indirect, between hypothalamus and the parasympathetic motor nuclei of the lower medulla.

Temporal pattern of HRP spread from an iontophoretic deposit site and description of a new HRP-gel implant method.

In the course of examining afferents to ventromedial hypothalamic (VMH) neurons using horseradish peroxidase (HRP), we needed to know how close to an iontophoretic deposit site neurons could be proved to be retrogradely labeled. In evaluating cells near but clearly outside HRP deposit sites visualized after a 24-hour survival period, for example, neurons which had been filled with HRP by somal or dendritic uptake could not be treated as retrogradely labeled and thus would not add to studies of intrahypothalamic connections. Rats were given standardized iontophoretic applications of HRP into VMH (continuous positive current 0.25 mu amp for 1 minute) and sacrificed after 5 or 15 minutes, 1, 4, 8, 12, or 24 hours in order to examine the pattern of HRP spread. The chromogen was tetramethylbenzidine. The volume of the application site visualized at 24 hours was less than 10% of maximum site size, which occurred at 1 hour. Since the cells located within the maximal spread boundary are candidates for nonretrograde labeling, HRP data on local connections obtained even from small iontophoretic deposits must be evaluated in the light of the demonstrated expansion and subsequent contraction of the application site. These results may also hold implications for the precision with which distant connections can be examined using the HRP retrograde method, as sites that appear discrete when visualized after 24-hour survival may have overlapped at shorter times post-iontophoresis. Incorporation of retrograde tracers into polyacrylamide gels provides an effective alternative to pressure injection or iontophoresis of aqueous tracer solutions. We describe a method for filling micropipettes with HRP-polyacrylamide gel. The pipettes are then implanted into brain sites to provide a confined pool of HRP. With postimplantation survival of 24 hour or longer, this method produces sites comparable in size to iontophoretic sites examined at 24 hours and results in improved retrograde labeling. Some results obtained with this method concerning the afferent connections of the dorsomedial hypothalamus are described.