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In the moth Manduca sexta 50% of the abdominal motor neurons and interneurons die during the first days after the adult emerges. Although the dying motor neurons innervate larval muscles that also die at this time, the death of the neurons has been shown to be a direct response to an endocrine signal, the decline in ecdysteroids that occurs at the end of metamorphosis. The response to the withdrawal of the hormone apparently requires new synthesis of RNA and protein, as actinomycin-D and cycloheximide can prevent neuronal death. This selective neuronal death probably results from changes in gene activation. The fixed spatial and temporal pattern of neuronal death, however, suggests that trophic interactions among neurons may also be involved. Current work is focused on the fate of a pair of motor neurons, the MN-12 cells, in the third abdominal ganglion. Isolation of this ganglion from the thoracic ganglia can prevent the death of these cells at the time when they would normally die in response to removal of ecdysteroids. The factors mediating this effect may act in concert with the ecdysteroid decline to specify the exact time of death for individual neurons.

Ce travail résulte de la collaboration de très nombreux chercheurs. Seuls les auteurs de la rubrique Physical and Genetic Mapping sont cités explicitement.; Here we report the genome sequence of the honeybee Apis mellifera, a key model for social behaviour and essential to global ecology through pollination. Compared with other sequenced insect genomes, the A. mellifera genome has high A1T and CpG contents, lacks major transposon families, evolves more slowly, and is more similar to vertebrates for circadian rhythm, RNA interference and DNA methylation genes, among others. Furthermore, A.mellifera has fewer genes for innate immunity, detoxification enzymes, cuticle-forming proteins and gustatory receptors, more genes for odorant receptors, and novel genes for nectar and pollen utilization, consistent with its ecology and social organization. Compared to Drosophila, genes in early developmental pathways differ in Apis, whereas similarities exist for functions that differ markedly, such as sex determination, brain function and behaviour. Population genetics suggests a novel African origin for the species A.mellifera and insights into whether Africanized bees spread throughout the New World via hybridization or displacement

▪ Abstract The past decade has produced an explosion of new information on the development, neuroanatomy, and possible functions of the mushroom bodies. This review provides a concise, contemporary overview of the structure of the mushroom bodies. Two topics are highlighted: the volume plasticity of mushroom body neuropils evident in the brains of some adult insects and a possible essential role for the γ lobe in olfactory memory.

You probably know that hard-working dung beetles keep the Serengeti Plain and cow pastures clean, but even most biologists are unaware of the efforts of another diligent group of beetles: the burying beetles of the genus Nicrophorus. Burying beetles play an important role in many ecosystems through their processing of small vertebrate carcasses. The reproductive cycle of such beetles is tied to the discovery of a recently deceased small animal (Scott, 1998; Trumbo, 1997). When beetles discover a carcass, they dig a hole beneath the body and cover it with dirt. They next remove the fur or feathers. The process is complete when the denuded corpse is covered with a layer of anal gland secretions. If more than two beetles find the carcass, males fight against males and females against females until a single pair is in control. Oviposition occurs within a day of the discovery of a carcass. The female lays her eggs in the soil; the larvae hatch about 4 days later and crawl to the carcass, which serves as nest and food source. For the first 2 days on the carcass, both parents feed the larvae regurgitated carrion and defend the carcass against other burying beetles. After this time, the larvae are able to feed themselves directly from the carcass, and the parents’ role is primarily defensive (Fetherston et al., 1990). The bout of parental behavior ends with the departure of the adult beetles from the nest: the female will remain until the larvae leave the carcass in preparation for pupation (6–8 days), while the male typically leaves a little sooner. A set of larvae can be raised successfully by a single parent as well as by a pair, with males able to compensate fully for the disappearance of the female after oviposition (Fetherston et al., 1994). Burying beetles therefore provide an uncommon example of extended, facultative biparental care in insects, of interest to entomologists, ecologists, and evolutionary biologists.

Insects have excellent color vision based on the expression of different opsins in specific sets of photoreceptive cells. Opsins are members of the rhodopsin superfamily of G-protein coupled receptors, and are transmembrane proteins found coupled to light-sensitive chromophores in animal photoreceptors. Diversification of opsins during animal evolution provided the basis for the development of wavelength-specific behavior and color vision, but with the exception of the recently discovered non-visual melanopsins, vertebrate and invertebrate opsins have generally been viewed as representing distinct lineages. We report a novel lineage of insect opsins, designated pteropsins. On the basis of sequence analysis and intron location, pteropsins are more closely related to vertebrate visual opsins than to invertebrate opsins. Of note is that the pteropsins are missing entirely from the genome of drosophilid flies. In situ hybridization studies of the honey bee, Apis mellifera, revealed that pteropsin is expressed in the brain of this species and not in either the simple or compound eyes. It was also possible, on the basis of in situ hybridization studies, to assign different long wavelength opsins to the compound eyes (AmLop1) and ocelli (AmLop2). Insect pteropsin might be orthologous to a ciliary opsin recently described from the annelid Platynereis, and therefore represents the presence of this vertebrate-like light-detecting system in insects.

We explored the neural basis of age- and task-related plasticity in circadian patterns of activity in the honeybee. To identify putative circadian pacemakers in the bee brain, we used antibodies against Drosophila melanogaster and Antheraea pernyi PERIOD and an antiserum to crustacean pigment-dispersing hormone (PDH) known to cross-react with insect pigment-dispersing factors (PDFs). In contrast to previous results from Drosophila, PDH and PER immunoreactivity (-ir) were not colocalized in bee neurons. The most intense PER-ir was cytoplasmic, in two groups of large neurons in the protocerebrum. The number of protocerebral PER-ir neurons and PER-ir intensity within individual cells were highest in brains collected during subjective night and higher in old bees than in young bees. These results are consistent with previous analyses of brain per mRNA in honeybees. Nuclear PER-ir was found throughout the brain, including the optic and antennal lobes. A single group of PDH-ir neurons (approximately 20/optic lobe) was consistently and intensely labeled at the medial margin of the medulla, independent of age or time of day. The processes of these neurons extended to specific neuropils in the protocerebrum and the optic lobes but not to the deutocerebrum. The patterns displayed by PER- and PDH-ir do not completely match any patterns previously described. This suggests that, although clock proteins are conserved across insect groups, there is no universal pattern of coexpression that allows ready identification of pacemaker neurons within the insect brain.

SUMMARY Three experiments were performed to determine why removal of the corpora allata (the glands that produce juvenile hormone) causes honey bees to fail to return to their hive upon initiating flight. In Experiment 1, the naturally occurring flights of allatectomized bees were tracked with radar to determine whether the deficit is physical or cognitive. The results indicated a physical impairment: allatectomized bees had a significantly slower ground speed than sham and untreated bees during orientation flights, but otherwise attributes such as flight range and area were normal. Flight impairment was confirmed in Experiment 2, based on observations of takeoff made in the field at the hive entrance. The allatectomized group had a significantly smaller percentage of flightworthy bees than did the sham and untreated groups. Experiment 3 confirmed the flight impairment in laboratory tests and showed that allatectomy causes a decrease in metabolic rate. Allatectomized bees had significantly lower metabolic rates than untreated and sham bees, while allatectomized bees receiving hormone replacement had intermediate values. These results indicate that allatectomy causes flight impairment, probably partly due to effects on metabolic rate. They also suggest that juvenile hormone plays an additional, previously unknown, role in coordinating the physiological underpinning of division of labor in honey bee colonies.

The extremely large number of insects and members of allied groups alive today suggests that molting--shedding of an old cuticle--may be one of the most commonly performed behaviors on our planet. Removal of an old cuticle in insects is associated with stereotyped, species-specific patterns of behavior referred to as ecdysis. It has been recognized for decades that the initiation of ecdysis is under hormonal control, but until recently many of the key peptides that regulate ecdysis were unknown. The report in 1996 of a new ecdysis-triggering hormone (ETH) sparked an era of significant advances in our understanding of the regulation of molting. This article summarizes the current model of peptide regulation of ecdysis, a model that is based on a positive feedback loop between ETH and a brain peptide, eclosion hormone. Then the relationship of these regulatory peptides to the neural circuitry that is the ultimate driver of the behavior are described. Because insects can undergo both status quo (larval-larval) and metamorphic (larval-pupal and pupal-adult) molts, differences in ecdysis behavior at different life stages are described and potential sources of these differences are identified. Most of the work described is based on studies of ecdysis in the hawkmoth, Manduca sexta, but results from studies of ecdysis in the fruit fly Drosophila melanogaster are also discussed.

Publisher Summary This chapter describes the proleg withdrawal reflex, the metamorphosis of leg motor neurons, gin trap reflex, and the initiation of wandering—all provide clear demonstrations of hormonal regulation of behavioral plasticity in insects. Substantial progress has been made because several different laboratories have concentrated their efforts on a single insect, the tobacco hornworm or Manduca sexta. Study of the neural bases of behavior in this insect has been facilitated by exploitation of identified cells in both neuroanatomical and electrophysiological studies. Instead of efforts focused on the identification of hormone target cells, the widespread distribution of the EcR gene product in the nervous system and associated structures has resulted in an emphasis on characterization of functional sites of hormone action within defined neural circuits. The field also retains a strong emphasis on behavioral assays.

The causes of the current global decline in honey bee health are unknown. One major group of hypotheses invokes the pesticides and other xenobiotics to which this important pollinator species is often exposed. Most studies have focused on mortality or behavioral deficiencies in exposed honey bees while neglecting other biological functions and target organs. The midgut epithelium of honey bees presents an important interface between the insect and its environment. It is maintained by proliferation of intestinal stem cells throughout the adult life of honey bees. We used caged honey bees to test multiple xenobiotics for effects on the replicative activity of the intestinal stem cells under laboratory conditions. Most of the tested compounds did not alter the replicative activity of intestinal stem cells. However, colchicine, methoxyfenozide, tetracycline, and a combination of coumaphos and tau-fluvalinate significantly affected proliferation rate. All substances except methoxyfenozide decreased proliferation rate. Thus, the results indicate that some xenobiotics frequently used in apiculture and known to accumulate in honey bee hives may have hitherto unknown physiological effects. The nutritional status and the susceptibility to pathogens of honey bees could be compromised by the impacts of xenobiotics on the maintenance of the midgut epithelium. This study contributes to a growing body of evidence that more comprehensive testing of xenobiotics may be required before novel or existing compounds can be considered safe for honey bees and other non-target species.

This article outlines the rationale for a molecular genetic study of social behavior, and explains why social insects are good models. Summaries of research on brain and behavior in two species, honey bees and fire ants, are presented to illustrate the richness of the behavioral phenomena that can be addressed with social insects and to show how they are beginning to be used to study genes that influence social behavior. We conclude by considering the problems and potential of this emerging field.

Juvenile hormone regulates metamorphosis in insects, and its effects on the nervous system during the larval-pupal transition have been studied primarily in the hawk moth, Manduca sexta. The effects of juvenile hormone on the nervous system of adult insects have been little studied. Elucidating the role of juvenile hormone during behavioral development in adult honey bees provides an opportunity to study hormone regulation of nervous system structure and function in an insect with a rich behavioral repertoire and social life. A worker honey bee typically lives 30-60 days. During this time, she performs a sequence of different tasks that sustain the colony. A striking behavioral transition typically occurs at about 3 weeks of age. At this time, worker bees stop performing within-hive tasks such as rearing brood and building comb and begin to forage outside the hive. This behavioral development is accompanied by a marked increase in the production of juvenile hormone. The mushroom bodies of the protocerebrum, the region of the insect brain most often associated with learning and memory, also undergo an internal reorganization during behavioral development. High titers of juvenile hormone and an increased volume of neuropil associated with the mushroom bodies are characteristic of the forager. Importantly, the time of the behavioral transition to foraging is not fixed. Individual bees can respond to changing colony or environmental conditions by accelerating or delaying the switch from within-hive tasks to foraging. For example, in the absence of older workers, some bees will undergo precocious development and may forage as early as 4 days of age. These workers also experience a precocious rise in juvenile hormone and an earlier reorganization of the mushroom bodies. Our current studies investigate the roles played by juvenile hormone and experience in shaping the mushroom bodies of the adult honey bee, and the relationship of these changes to the bee's ability to forage successfully. It is proposed that juvenile hormone may mediate neural plasticity in the brains of adult honey bees to support the demanding cognitive task of foraging.

What can we learn from the brain of a single member of a species? If the brain is that of a remipede, the focus of a report by Fanenbruck et al. (1) in this issue of PNAS, the answer is something new about arthropod phylogeny. The Remipedia, discovered in 1979, are crustaceans found deep in the waters of coastal caves (2, 3). To date, twelve species of remipedes are known: one from a single cave in Western Australia and the others from the Caribbean. The remipede body is divided into a head and an elongated trunk with up to 32 segments. The trunk segments feature paddle-like appendages that give the taxon its name (“oar feet”). Remipedes show adaptations to life in darkness, including the absence of pigmentation and eyes, but like other crustaceans they have two pairs of antennae. The evolutionary history of these rare arthropods is uncertain. In their study of the brain of Godzilliognomus frondosus , Fanenbruck et al . (1) provide a fresh look at remipede phylogeny. Their neuroanatomical studies suggest that the Remipedia are most likely part of a larger clade that includes the Malacostraca and the Hexapoda, two arthropod groups well known for their complex brains. This result is surprising because, at first inspection, the simplicity of the remipede body plan suggests that this group provides a glimpse of untagmatized ancestors with numerous unspecialized appendages and should be placed in a basal position in the crustacean lineage (4). This view, based on gross morphology, is contradicted by molecular analyses suggesting that the remipedes are a sister group to the hexapods (six-legged arthropods, including insects) (5, 6). These studies were based on comparisons of sequences for RNA polymerase II and elongation factors 1 and 2. The results not only elevated the Remipedia from a …

The intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta, participate in the emergence behavior of the adult moth and then die during the subsequent 30 hours. In addition, several populations of interneurons and uniquely identified motor neurons also die after adult emergence. The trigger for all of these deaths is a decline in the circulating titer of the insect molting hormone 20-hydroxyecdysone. The ability of the muscles and neurons to die requires de novo gene expression. A differential hybridization screen of a "condemned" ISM cDNA library permitted the isolation of clones encoding four new up-regulated mRNAs. On sequencing, one of these recombinants was found to encode apolipophorin III (apoLp-III), a component of lipophorin, the major hemolymph lipoprotein of insects, previously shown to be synthesized in fat body. Although apoLp-III mRNA and protein were expressed at all stages of ISM development, levels of both molecules were dramatically elevated with the commitment of the cells to die. When ISM cell death was delayed by injection of 20-hydroxyecdysone, expression of apoLp-III at both the RNA and protein levels was markedly reduced at the normal time of cell death. Immunocytochemistry demonstrated that apoLp-III protein was abundantly expressed in the cytoplasm of dying muscles, interneurons, and identified motor neurons at the time of cell death. Apolipoproteins I and II, required components of lipophorin, were not expressed at detectable levels in the muscles or neurons. Furthermore, Western blots of native gels suggest that apoLp-III was not associated with any other proteins. These data suggest that apoLp-III has activities independent of lipid transport that may play a role in programmed cell death. ApoLp-III joins apolipoproteins E and J (clusterin, sulfated glycoprotein-2) as a group of proteins that function in both lipid transfer and cell death.

A restrained honey bee can be trained to extend its proboscis in response to the pairing of an odor with a sucrose reward, a form of olfactory associative learning referred to as the proboscis extension response (PER). Although the ability of flying honey bees to respond to visual cues is well-established, associative visual learning in restrained honey bees has been challenging to demonstrate. Those few groups that have documented vision-based PER have reported that removing the antennae prior to training is a prerequisite for learning. Here we report, for a simple visual learning task, the first successful performance by restrained honey bees with intact antennae. Honey bee foragers were trained on a differential visual association task by pairing the presentation of a blue light with a sucrose reward and leaving the presentation of a green light unrewarded. A negative correlation was found between age of foragers and their performance in the visual PER task. Using the adaptations to the traditional PER task outlined here, future studies can exploit pharmacological and physiological techniques to explore the neural circuit basis of visual learning in the honey bee.

A decline in circulating 20-hydroxyecdysone permits the emergence of the adult Manduca sexta moth; this endocrine signal also triggers the death of approximately half of the neurons in the unfused abdominal ganglia of the moth central nervous system. This programmed death of neurons was markedly reduced by treatment with either actinomycin D (an RNA synthesis inhibitor) or cycloheximide (a protein synthesis inhibitor). Similar results were found after addition of these agents to ventral nerve cord cultures. The effectiveness of these treatments in delaying or blocking neuronal death depended upon their time of administration relative to the normal time of post-emergence death in the particular motoneuron under study: late-dying neurons, for example, could still be saved by these treatments even after early-dying neurons had already initiated degeneration. In both intact moths and cultured ventral nerve cords, the ability of actinomycin D to prevent neuronal death waned at the same time at which replacement of the steroid hormone could no longer block neuronal death. This suggests that the steroid commitment point represents the time at which the genes that mediate cell death are transcribed. Cycloheximide remained effective in delaying or blocking neuronal death until shortly before the onset of degeneration, suggesting that ongoing protein synthesis is essential for the initiation of the degeneration response.

The mushroom bodies in the protocerebrum are believed to be the structures of the insect brain most closely associated with higher-order sensory integration and learning. Drosophila melanogaster mutants with olfactory learning deficits have anatomically abnormal mushroom bodies or altered patterns of gene expression in mushroom body neurons. In addition, anatomical reorganization of the mushroom bodies occurs in adult flies, and possibly in adult honeybees; disturbance of electrical activity in this region disrupts memory formation in honeybees. Little is known, however, about the relationship of naturally occurring anatomical changes in the mushroom bodies to naturally occurring behavioural plasticity. We now report that age-based division of labour in adult worker honeybees (Apis mellifera) is associated with substantial changes in certain brain regions, notably the mushroom bodies. Moreover, these striking changes in brain structure are dependent, not on the age of the bee, but on its foraging experience, thus demonstrating a robust anatomical plasticity associated with complex behaviour in an adult insect.

Publisher Summary This chapter deals with the use of invertebrates to study cellular and molecular aspects of neuronal death. The identified neurons in these animals that are well known for their stereotyped morphologies and patterns of connectivity may likewise have stereotyped, predictable fates. Thus, one can examine not only the changes that occur after degeneration begins, but also the events that precede death. Much of this work has been done on insects, in which neuronal death plays a prominent role both during embryogenesis and at metamorphosis. The neurons that die during the latter period include both immature cells and mature, functioning neurons that are used during larval life but then discarded. The occurrence of cell death as part of metamorphosis means that these events have come under the control of the hormones that regulate metamorphosis. Besides being able to manipulate the timing of cell death by endocrine manipulations, hormones have also provided useful tools for probing cellular interactions in the degeneration response.

The emergence of the adult Manduca sexta moth is followed by the loss of almost half of this insect's abdominal motoneurons and interneurons (Truman, 1983). This programmed cell death completes the transformation of the nervous system of the caterpillar into that of the moth. The death of these neurons has been previously shown to be a response to an endocrine signal: the decline in ecdysteroids that occurs at the end of metamorphosis (Truman and Schwartz, 1984). Our current research is focussed on the regulation of the fate of a pair of identified motoneurons, the MN-12 cells, in the third abdominal ganglion. Isolation of this ganglion from anterior parts of the nervous system can prevent the death of these cells at the time when they would normally die in response to the decline in ecdysteroids. Transection of the ventral nerve cord at various levels revealed that the source of this regulatory "death signal" is the fused pterothoracic ganglion and that it is transmitted via the interganglionic connectives. We hypothesize that the factors mediating this effect may act in concert with the ecdysteroid decline to specify the exact time of death for individual neurons.

The afferent neural connections of the ventromedial nucleus of the rat hypothalamus (VMH) have been studied in detail using three horseradish peroxidase (HRP) application methods: HRP crystal implants, HRP-gel implants, and iontophoretic deposition of the enzyme. Examination of the cases in which the retrograde tracer was best confined to various subdivisions of the nucleus revealed that the septal area projects only to the ventrolateral VMH, and that the medial preoptic area, rostral lateral hypothalamus, and the ventral subiculum project mainly to the ventrolateral VMH. Thus, the subdivision of the VMH that contains the highest density of estradiol-concentrating neurons (Morrell et al. 1986) receives a larger set of inputs than the rostral and central parts of the nucleus. The central subdivision receives a more restricted set of projections than either the medial or the lateral regions. These studies suggest that there may be partial anatomical segregation of neural inputs to the various subdivisions of the VMH.

Intracerebroventricular (i.c.v.) infusions of oxytocin (OXY) induce short-latency maternal behavior in estrogen-primed virgin rats. To investigate if brain OXY might have a role in the onset of maternal behavior at parturition, we have used both antisera to OXY and an analog antagonist of OXY, d(CH2)5-8-ornithine-vasotocin, to reduce the activity of endogenous OXY in a pregnancy-terminated preparation which yields reliable short-latency maternal behavior. Sprague-Dawley rats with lateral ventricle cannulae were ovariectomized and hysterectomized on day 16 of gestation; maternal behavior was stimulated by a s.c. injection of estradiol benzoate (EB). Effects of the i.c.v. infusion of antisera to OXY or of the i.c.v. infusion of d(CH2)5-8-ornithine-vasotocin on the latency to respond to pups were tested by presenting pups 48 h after surgery and EB treatment. Behavioral observations were made for the next 5 h and periodically over the next 5 days. Groups receiving either the antisera to OXY or the analog antagonist had significantly longer latencies to respond to pups than did control groups. In a separate experiment, i.c.v. infusion of d(CH2)5-8-ornithine-vasotocin was shown to have no effect on the performance of maternal behavior in lactating rats 5 days postpartum. These results suggest that OXY may have a role in promoting short-latency maternal behavior in steroid-primed female rats, but that it is probably not involved in sustaining this behavior during lactation.

In order to localize the neural substrate for estrogen induction of increases in activity, bilateral implants of dilute estradiol (in concentrations of 1:10, 1:100, and 1:250) were made into the medial preoptic area (n=5), the anterior hypothalamus (n=8), the ventromedial hypothalamus (n=6), and the posterior hypothalamus (n=4) of ovariectomized female rats housed in running wheels. The same animals were also tested for open field activity and lordosis. The preoptic area was the most effective site tested for estrogenic stimulation of running wheel activity, with 3 5 animals responding consistently to estradiol application. 3 8 animals with cannulae in the anterior hypothalamus (AHA) responded with increased in activity measured in the running wheel on at least two out of three estradiol treatments. No animals with cannula placements posterior to the AHA exhibited consistent running wheel responses to estradiol. No estrogen-induced increase in open field activity was found, regardless of cannula location or concentration of estradiol; however, there was a tendency for the number of squares entered during the later tests to decrease in the ventromedial hypothalamic nucleus group and in the subgroup of animals not showing estrogen-sensitive wheel running in the anterior hypothalamic area group. Only the ventromedial hypothalamic estradiol treatments stimulated lordosis behavior. These results support earlier reports suggesting that the medial preoptic area (and possibly the adjacent anterior hypothalamic area) is the critical brain site for estrogenic stimulation of running wheel activity. Since estradiol treatments effective in stimulating running in an activity wheel did not increase locomotion in the open field, it is possible that estrogen effects on open field measures of activity are regulated separately.

The steroid hormone 20-hydroxyecdysone regulates many aspects of nervous system development in the moth Manduca sexta, including stage-specific neuronal morphology and stage-specific neuronal death. We have used steroid hormone autoradiography to study the distribution of cells that concentrate ecdysteroids in the ventral nervous system of this insect. The ligand was [3H]-ponasterone A, a bioactive phytoecdysone. Tissue was examined from three stages of development: the end of larval life (first day of wandering), the end of metamorphosis (pharate adult), and 4-day-old adults. In the abdominal ganglia of wandering larvae and pharate adults, a subset of neurons including both motoneurons and interneurons exhibited a nuclear concentration of radiolabeled hormone. The pattern of binding was reproducible but stage-specific, with a greater proportion of neurons showing binding in the larvae than in pharate adults. No labeled neurons were found in abdominal ganglia from mature (4-day-old) adults. In the case of the pharate adult ganglia, the ecdysteroid receptor content of specific, identified motoneurons was determined. These results are discussed in light of the responses of these neurons to physiological changes in levels of circulating ecdysteroids.

This study was designed to test whether intracerebroventricular (icv) administration of oxytocin can shorten the latency of estrogen-treated virgin rats to respond to foster pups with maternal care. A secondary goal was a description of the behavioral effects of icv oxytocin in rats. Forty-eight hours before pup presentation female rats (Sprague-Dawley strain, obtained from Zivic-Miller Laboratories, Inc.) with chronic lateral ventricle cannulae were ovariectomized and treated with 100 micrograms/kg body wt estradiol benzoate (EB). Immediately before pup presentation either 400 ng oxytocin or the normal saline vehicle was infused. A second control group of noncannulated animals received the same surgery, EB treatment, and behavior tests without any icv infusion. Maternal behavior was scored on a scale from 0 to 5, one point each being given for licking pups, carrying pups, grouping pups, crouching over pups, and nest building. At the end of 1 hr of pup exposure 65% of the oxytocin-infused animals received scores of 4 or 5, while only 25% of the control animals attained these scores. Maternal behavior appeared as a unit in both spontaneously maternal and oxytocin-treated animals, with fewer than 10% in either group receiving scores of 1, 2, or 3. The oxytocin group also spent significantly more time grooming. In a second experiment in which oxytocin or normal saline was administered icv to oil-treated animals, this grooming effect was shown to be estrogen independent while the induction of maternal behavior required prior treatment with estrogen. Finally, a slight, transient hyperthermic effect of oxytocin on body temperature was observed in this test paradigm. These data suggest a role for oxytocin, acting with estrogen, in the onset of maternal behavior in rats.

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.

The axonal projections of cell groups containing the most dense collections of steroid hormone concentrating cells have been demonstrated with retrograde neuroanatomical tracing methods. Horseradish peroxidase revealed large numbers of neurons in ventrolateral ventromedial nucleus (VL-VM) which project to dorsal midbrain. Wheat germ agglutinin (immunocytochemical recognition method) revealed large numbers of neurons in medial basal hypothalamus (MBH) and particular subdivisions of paraventricular nucleus (PVN) that project to dorsal caudal medulla or spinal cord. Fluorescent dyes revealed that many preoptic area (POA), anterior hypothalamic (AHA), and bed nucleus of the stria terminalis (BNST) neurons project to ventral tegmental area of Tsai (VTA). Also many neurons in POA and BNST project to amygdala. A method which enabled simultaneous demonstration of the steroid binding capacity and axonal projections of neurons in the same tissue section revealed that 26-36% estradiol (E2) concentrating cells in VL-VM project to dorsal midbrain. E2 concentrating neurons in POA and BNST project to amygdala and E2 concentrating POA neurons project to VTA. These neurons, which send their axons to cell groups located in different brain regions, are probably under the genomic-regulatory influence of E2. Using a method which allows simultaneous demonstration of peptide content and steroid hormone concentrating capacity of cells, many oxytocin-neurophysin and vasopressin-neurophysin containing magnocellular neurons in the caudal PVN were found to concentrate E2. About 4% of the beta-endorphin and about 6% of the dynorphin containing neurons in the MBH concentrate E2. In contrast, virtually none (less than 0.2%) of the LHRH containing hypothalamic neurons concentrate E2.

This chapter will survey current ideas on the physiological bases of maternal behavior in the rat. Maternal behavior is, of course, a characteristic of all mammals, and ethological descriptions of maternal care are on record for many species (Lehrman, 1961; Klopfer, McGeorge, & Barnett, 1973). However, only a few species have been the subjects of laboratory investigations of mother-young interactions. Included among these are the rat, mouse, hamster, gerbil, rabbit, and sheep, with the rat being the best studied in terms of physiological mechanisms. This fact has determined this chapter’s emphasis, but one should not assume that the rat can serve as a general model for the regulation of maternal behavior in mammals. Striking interspecies differences, both in behavior and hormone profiles, have already been described; the significance of these differences is not yet clear.

Intracerebroventricular (ICV) infusion of the peptide hormone oxytocin has been previously reported to induce the performance of short-latency maternal behavior (less than one hour of exposure to foster pups required) in estrogen-treated, ovariectomized virgin rats. Tests for the effect of ICV oxytocin in maternal behavior latency have included transfer of animals from their home cage to a larger test cage one to two hours before oxytocin infusion. The importance of this test feature on peptide-induced short-latency maternal behavior was evaluated by varying the duration of the pre-test cage habituation. The responses of ovariectomized, estrogen-primed Zivic-Miller Sprague-Dawley rats housed in the test cages one week, two hours, or 0 hours before oxytocin or saline infusion were compared. It was found that only the rats given two hours of pre-test cage habituation responded to ICV oxytocin treatment with short-latency maternal behavior. This result is discussed with regard to the failure of other investigators to elicit short-latency maternal behavior with oxytocin. Possible neuroendocrine mechanisms for the interaction of degree of environmental novelty with oxytocin-induced behavior are considered.

Estrogen implanted directly into the medial preoptic region of pregnant Charles River Sprague-Dawley rats hysterectomized and ovariectomized on Day 16 of gestation mimics the effects of systemic estrogen treatment at this time by reducing the latency to respond to foster pups with maternal behavior (Numan, Rosenblatt, and Komisaruk, 1977). The present report describes the pup-directed responses of ovariectomized, nulliparous Zivic-Miller Sprague-Dawley rats that received bilateral medial preoptic implants of either cholesterol (n = 11) or estradiol diluted 1:10 with cholesterol (n = 11). Two days after treatment these animals were housed with three foster pups: their responsivity to pups and quality of nests built were then assessed, at first hourly and then daily. Rats receiving intracranial estradiol required significantly shorter exposures to pups than did cholesterol-treated animals before initiating carrying and grouping of 3 dispersed pups in a maternal nest during a 15-min test. On other measures, however, the groups did not differ (e.g., proportion grouping pups overnight, time required to complete retrieval of pups to the nest, time required to rebuild a disrupted nest). Animals treated with cholesterol and animals with estradiol implants did not differ in uterine weight at the time of sacrifice, suggesting that estrogen did not leak, even from this well-vascularized implant site, into the circulation. Thus, as in the pregnant animal, the facilitating effects of estrogen on maternal behavior can be mediated through the medial preoptic region; however, the effects were evident only when a test requiring retrieval of several pups within an arbitrarily short interval was given.

Fluorescent dye retrograde tracing was combined with steroid hormone autoradiography to study the midbrain projections of the estrogen-concentrating neurons in the preoptic region of the rat brain. Microinjections of the dyes DAPI, true blue, or a mixture of DAPI and primuline were made into the ventral tegmental area and into the midbrain central gray of ovariectomized, adrenalectomized 2-3-month-old female rats; 3 or 4 days later these animals were injected with [3H]estradiol; the brains were then processed for autoradiography. After exposures of from 3 to 12 months, the autoradiograms were developed and examined for reduced silver grains under cell nuclei (indicating binding of [3H]estradiol) and retrogradely transported fluorescent dye in the cytoplasm (indicating an efferent projection to the midbrain). Numerous [3H]estradiol-concentrating neurons in the medial preoptic region were found to send their axons to the medial midbrain. The largest numbers of estrogen target neurons that were afferent to the ventral tegmental area and to the midbrain central gray were found in the medial preoptic nucleus, in the surrounding medial preoptic area, and in the ventral bed nucleus of the stria terminalis. Double-labeled neurons were also identified in the preoptic suprachiasmatic area, in the lateral preoptic area, and in the rostral anterior hypothalamic area. Thus, a subset of the gonadal steroid target cells of the preoptic region have long projections to the medial midbrain, and a subset of the medial preoptic neurons that project to the ventral tegmental area and to the midbrain central gray concentrate estrogen. Behaviors (for example, maternal behavior, male copulatory behavior, and wheel-running) that are regulated by estrogen action in the medial preoptic region may be controlled by the direct estrogen-sensitive pathway to the medial midbrain revealed in this study.