[DLys(6)]-luteinizing hormone releasing hormone-curcumin conjugate inhibits pancreatic cancer cell growth in vitro and in vivo.

Pancreatic ductal adenocarcinomas are invariably lethal, and developing effective treatments that have minimal side effects is a challenge. Previous studies from our laboratory have shown that conjugates of cell membrane disrupting lytic peptides and luteinizing hormone releasing hormone (LHRH) target and destroy human prostate and breast cancer cells in xenografts in the nude mouse model (Hansel et al., Mol Cell Endocrinol 2007;260-262:183-9; Hansel et al., Mol Cell Endocrinol 2007;269:26-33), which express LHRH receptors. The objectives of our study were to synthesize a bioconjugate of LHRH analog ([DLys(6)]-LHRH) and a dietary microchemical (curcumin) and test the hypothesis that [DLys(6)]-LHRH-curcumin targets and inhibits pancreatic cancer cell growth in vitro and in vivo. In in vitro studies, we determined by confocal microscopy, flow cytometry analysis and reverse transcriptase-polymerase chain reaction that MIAPaCa-2, Panc-1 and BxPC-3 pancreatic cancer cell lines express LHRH receptors. [DLys(6)]-LHRH-curcumin inhibited cell proliferation of pancreatic cancer cell lines and induced apoptotic cell death (p < 0.05). Apoptosis was induced by cleavage of polyadenosine-5'-diphosphate-ribose-polymerase and caspase-3. The activity of [DLys(6)]-LHRH-curcumin was equal to free curcumin at equimolar concentrations in vitro. Unlike curcumin itself, the [DLys(6)]-LHRH-curcumin conjugate is water soluble which allows its intravenous administration. In two in vivo studies, [DLys(6)]-LHRH-curcumin given intravenously caused a significant (p < 0.01) reduction in tumor weights and volumes, and free curcumin given by gavage at an equal dose failed to cause a significant reduction in tumor weights and volumes in the nude mouse pancreatic cancer model. [DLys(6)]-LHRH-curcumin treatment enhanced apoptosis compared to [DLys(6)]-LHRH and vehicle-treated controls in tumor tissue. In conclusion, [DLys(6)]-LHRH-curcumin may be useful in treating pancreatic cancer.

Improved fidelity of thermostable ligases for detection of microsatellite repeat sequences using nucleoside analogs.

Microsatellite repeats consisting of dinucleotide sequences are ubiquitous in the human genome and have proven useful for linkage analysis, positional cloning and forensic identification purposes. In this study, the potential of utilizing the ligase detection reaction for the analysis of such microsatellite repeat sequences was investigated. Initially, the fidelity of thermostable DNA ligases was measured for model dinucleotide repeat sequences. Subsequently, the effect of modified oligonucleotides on ligation fidelity for dinucleotide repeats was determined using the nucleoside analogs nitroimidazole, inosine, 7-deazaguanosine and 2-pyrimidinone, as well as natural base mismatches. The measured error rates for a standard dinucleotide template indicated that the nitroimidazole nucleoside analogs could be used to increase the fidelity of ligation when compared to unmodified primers. Furthermore, use of formamide in the ligation buffer also increased ligation fidelity for dinucleotide repeat sequences. Using ligation-based assays to detect polymorphic alleles of microsatellite repeats in the human genome opens the possibility of using array-based typing of these loci for human identification, loss-of-heterozygosity studies and linkage analysis.

Universal DNA microarray method for multiplex detection of low abundance point mutations.

Cancers arise from the accumulation of multiple mutations in genes regulating cellular growth and differentiation. Identification of such mutations in numerous genes represents a significant challenge in genetic analysis, particularly when the majority of DNA in a tumor sample is from wild-type stroma. To overcome these difficulties, we have developed a new type of DNA microchip that combines polymerase chain reaction/ligase detection reaction (PCR/LDR) with "zip-code" hybridization. Suitably designed allele-specific LDR primers become covalently ligated to adjacent fluorescently labeled primers if and only if a mutation is present. The allele-specific LDR primers contain on their 5'-ends "zip-code complements" that are used to direct LDR products to specific zip-code addresses attached covalently to a three-dimensional gel-matrix array. Since zip-codes have no homology to either the target sequence or to other sequences in the genome, false signals due to mismatch hybridizations are not detected. The zip-code sequences remain constant and their complements can be appended to any set of LDR primers, making our zip-code arrays universal. Using the K- ras gene as a model system, multiplex PCR/LDR followed by hybridization to prototype 3x3 zip-code arrays correctly identified all mutations in tumor and cell line DNA. Mutations present at less than one per cent of the wild-type DNA level could be distinguished. Universal arrays may be used to rapidly detect low abundance mutations in any gene of interest.

Dendritic vacuolization in the central nervous system of rats after long-term voluntary consumption of ethanol.

Adult male rats were housed in a colony environment for six months, with ad lib access to anise-flavored 10% ethanol in water. Animals were then removed from the colony, and their consumption of alcohol during a period in isolated housing was measured. Individual rats were scored as high, moderate, and low consumers. Animals from each scoring category were killed for light and electron microscopic study of central nervous system tissue. High consumers frequently displayed varicose distortions of the dendritic profiles, with internal membranous vesicles. Such abnormalities were rarely found in dendrites of low-ethanol-consuming colony mates. The dendritic vacuoles often appeared empty and membrane-limited. Some vacuoles contained membranous inclusions. Dendrites which displayed electron-lucent cavities without membranous limits or contents were also found. Some invaginations of dendritic membranes were identified. The possible sequential relationship between these forms of dendritic alterations could not be determined. Some neuron cell bodies displayed vacuolar inclusions as well. Dendritic and somatic abnormalities were found in cerebral and cerebellar cortices, hippocampus, mammillary bodies, and the periaqueductal gray matter of the brain stem.

Altered metabolic activity in the cerebral cortex of rats exposed to ketamine.

Uptake of the metabolic marker, [3H]2-deoxy-D-glucose (2DG) was compared in rats given subanesthetic (50--100 mg/kg, i.p.) or anesthetic (200 mg/kg, i.v.) doses of ketamine with that in normal, unanesthetized rats. All doses of ketamine caused a relative increase of 2DG labeling in limbic regions, including the hippocampus, dentate gyrus, and cingulate, piriform, and entorhinal cortices. Striking 2DG-dense zones were confined to the molecular layer in the hippocampus, dentate gyrus, and entorhinal cortex. Subanesthetic doses of ketamine produced a relative reduction of 2DG uptake in layers I--IV of granular somatosensory cortex while sparing uptake in layer Va; therefore, the peak of dense uptake shifted from layer IV to layer Va. In regions of the somatosensory cortex which display a dysgranular layer IV, vertical columns of relatively dense 2DG uptake extended through all cortical layers. Columns of 2DG label also occurred outside of S1, in visual and auditory areas. In the primary visual cortex, this dose of ketamine decreased 2DG uptake relative to secondary visual cortex. Alteration of 2DG uptake in various cortical regions might be the consequence of a ketamine-induced activation of specific neuronal pathways with special neurochemical features. During subanesthetic ketamine administration, peak 2DG uptake shifts from cortical layer IV, which receives specific thalamocortical input, to layer Va, which receives projections via intrinsic cortical circuits. The ketamine-induced shift in the laminar focus of sensory cortical metabolism may reflect a functional disconnection from peripheral sensory input and/or enhanced internal (corticocortical) processing.(ABSTRACT TRUNCATED AT 250 WORDS)

Tritiated 2-deoxy-D-glucose: a high-resolution marker for autoradiographic localization of brain metabolism.

The technique for autoradiographic localization of 2-deoxy-D-glucose (2DG) uptake has become a useful method for observing alterations of functional brain activity resulting from experimental manipulation. Autoradiographic resolution is improved using tritiated ([3H]) rather than carbon-14 ([14C)]2DG, due to the lower energy and shorter path of tritium emissions. In addition, lower 2DG uptake by white matter relative to gray matter is exaggerated in the [3H]2DG autoradiographs due to the greater absorption of tritium emissions by lipids. Using [3H]2DG, it is possible to observe differential metabolic labeling in various individual nuclei or portions of nuclei that is unresolvable using [14C]2DG in the awake, normal animal. Heterogeneous patterns of 2DG uptake seen only with [3H]2DG are found in the nucleus accumbens, the anterior portion of the basolateral nucleus of the amygdala, specific nuclei of the inferior olivary complex, various hypothalamic regions, and a region straddling the border of the medial and lateral habenular nuclei. The lamination of differential 2DG uptake in the hippocampus is better localized using [3H]2DG. Autoradiographic resolution of labeled 2DG is further improved when the brain is perfused prior to frozen sectioning, due perhaps to selective fixation and retention of intracellular labeled 2-deoxy-glycogen. A series of [3H]2DG autoradiographs are presented together with views of the Nissl-stained sections that produced the autoradiographs.

Cocaine abstinence following chronic treatment alters cerebral metabolism in dopaminergic reward regions. Bromocriptine enhances recovery.

2-[14C]deoxyglucose autoradiography was used to determine local cerebral glucose utilization (lCGU) in rats following chronic cocaine treatment and subsequent abstinence. lCGU was examined in 43 discrete brain regions in animals which had received daily injections of cocaine for 14 days (10 mg/kg) followed by 3 days of saline or bromocriptine (10 mg/kg) treatment. Cocaine abstinence following chronic treatment significantly reduced lCGU in several regions including mesocorticolimbic structures such as ventral tegmental area, medial prefrontal cortex, and nucleus accumbens (NAc). Within the NAc, however, only the rostral pole showed significant reduction. In contrast, when bromocriptine treatment accompanied abstinence, lCGU was no longer reduced in mesocorticolimbic and most other regions, implying that metabolic recovery was enhanced by bromocriptine treatment during early abstinence following chronic cocaine treatment. These data suggest that cerebral metabolism is decreased during cocaine abstinence following chronic treatment in critical brain regions, and that this alteration can be prevented by treatment with direct-acting dopamine agonists such as bromocriptine.

The disruptive effect of cocaine on prepulse inhibition is prevented by repeated administration in rats.

These experiments tested the hypothesis that pretreatment with a behaviorally sensitizing regimen of cocaine alters the ability of cocaine to disrupt prepulse inhibition (PPI). Male Sprague-Dawley rats were treated with cocaine (30 mg/kg, i.p.) or saline vehicle for seven consecutive days followed by challenge treatment seven days later. Repeated cocaine treatment produced sensitization of stereotyped activity. Cocaine challenge following repeated vehicle treatment significantly reduced PPI, but this effect was completely abolished by repeated cocaine treatment. These data suggest that neuroadaptation following repeated treatment might prevent PPI disruption caused by psychomotor stimulants.

Autoradiographic localization of dopamine D1 and D2 receptors in rat nucleus accumbens: resistance to differential rearing conditions.

The radioligands [3H]SCH 23390 and [3H]spiroperidol were used to label dopamine D1 and D2 receptors, respectively, in rat brain slices. Striatal sections were incubated in one of various concentrations of radioligand in the presence or absence of (+)-butaclamol and the resulting labeling was determined by liquid scintillation spectrometry. Scatchard analyses of the data revealed KD values of 1.18 nM for D1 receptors and 0.33 nM for D2 receptors. Tissue sections taken from the entire rostrocaudal extent of the nucleus accumbens, as well as other brain regions, were then processed for autoradiographic analysis of D1 and D2 receptors using a radioligand concentration equal to 1.5 X KD. After apposing the slices to 3H-sensitive film, topographical differences among brain areas were analysed using a quantitative densitometry system which determined the absolute amount of ligand binding relative to calibrated optical density standards. The nucleus accumbens exhibited a rostral-to-caudal density gradient for both D1 and D2 receptors. For D1 receptors, the density was similar across most of the nucleus accumbens, although the most caudal portion examined had a lower density than rostral portions. In contrast, the density of D2 receptors exhibited a more gradual gradient across the entire rostrocaudal extent of the nucleus accumbens. There was no significant rostrocaudal density gradient of either D1 or D2 receptors in either the olfactory tubercle or caudate-putamen in the same tissue sections. A lateral-to-medial gradient of D2 receptors was also present in the nucleus accumbens. That is, while there was no difference in the density of D1 receptors between the lateral core and medial shell subdivisions, the shell had a lower density of D2 receptors than did the core. The density of D1 and D2 receptors in the mesolimbic and nigrostriatal systems was compared in groups of animals raised from 30 to 60 days of age in an impoverished condition, a group-caged condition or an enriched condition. While the brain weight of enriched condition animals was higher than impoverished condition animals, there were no significant differences in the density of D1 or D2 receptors among the different groups. Apparently, the densities of D1 and D2 receptors in the brain are resistant to differential rearing conditions.

Reduction of zif268 messenger RNA expression during prolonged withdrawal following "binge" cocaine self-administration in rats.

Cocaine self-administration increases dopamine efflux and neuronal activity in the mesocorticolimbic dopamine system compared with experimenter-administered cocaine. Following a prolonged cocaine self-administration binge, dopamine efflux in the nucleus accumbens is attenuated and behaviors emerge that are indicative of anhedonia and anxiety. The neuronal correlates of these behavioral and neurochemical effects of a cocaine binge were assessed using in situ hybridization histochemistry to detect changes in zif268 messenger RNA expression. Rats were fitted with intravenous catheters; one group was trained to self-administer cocaine (0.5mg/injection), then allowed continuous access to cocaine during a 16h binge, while yoked animals received either saline or cocaine according to the same schedule. Measurement of tactile startle responses and ultrasonic distress calls either immediately after termination of cocaine access or one or 14 days later confirmed peak withdrawal at 24h after the binge. The level of zif268 messenger RNA was lower upon termination of cocaine self-administration than in both yoked treatment groups in the ventral tegmental area and hippocampus. In contrast, zif268 messenger RNA expression increased in the periaqueductal gray matter one day after termination of passive cocaine treatment, coincident with enhanced expression of ultrasonic vocalizations. Zif268 messenger RNA expression decreased over time in the nucleus accumbens core and infralimbic cortex, with reduced expression observed in the nucleus accumbens core, caudatoputamen, hippocampus and amygdala 14 days after termination of cocaine self-administration. The results suggest that withdrawal following a cocaine self-administration binge produces a long-lasting reduction of constitutive zif268 messenger RNA expression in mesocorticolimbic brain regions related to the nucleus accumbens. The relatively greater effect in animals that self-administered cocaine implies a relationship of certain regional responses to behavioral conditioning.

Long-term behavioral and neuronal cross-sensitization to amphetamine induced by repeated brief social defeat stress: Fos in the ventral tegmental area and amygdala.

Repeated exposure to stress induces cross-sensitization to psychostimulants. The present study assessed functional neural activation during social defeat stress-induced sensitization to a subsequent amphetamine challenge. Social defeat stress was induced in intruder rats during short confrontations with an aggressive resident rat once every third day during the course of 10 days. Rats received d-amphetamine injections (1 mg/kg, i.p.) 17 or 70 days after the first social defeat stress exposure. Amphetamine administration induced a significantly higher frequency of locomotor activity in stressed animals than in handled control rats, which was still evident 2 months after the last social stress exposure. Immunohistochemistry for Fos-like proteins was used to detect activated neural profiles in the striatum, nucleus accumbens (NAc), prefrontal cortex, amygdala, and ventral tegmental area (VTA). Repeated social defeat stress significantly increased Fos-like immunoreactive (Fos-LI) labeling 17 days after the start of stress exposure in the prelimbic and infralimbic cortical regions, NAc shell and core, medial, central and basolateral amygdala, and VTA, which probably represented the expression of chronic Fos-related antigens. Amphetamine augmented stress-induced Fos-LI labeling 17 days after the first stress episode in the dorsal striatum, NAc core, and medial amygdala, reflecting a cross-sensitization of Fos response. Amphetamine challenge 70 days after social stress exposures revealed sensitized Fos-LI labeling in the VTA and the amygdala. These data suggest that episodes of repeated social stress induce a long-lasting neural change that leads to an augmented functional activation in the VTA and amygdala, which might represent a neurobiological substrate for long-lasting cross-sensitization of repeated social defeat stress with psychostimulant drugs.

Estrogenic regulation of proenkephalin mRNA expression in the ventromedial hypothalamus of the adult male rat.

Endogenous opioid peptides and their receptors are regulated by gonadal steroid hormones in the rat hypothalamus. Recent evidence suggests that gonadal steroids are capable of regulating the expression of proenkephalin (PE) mRNA in the ventromedial hypothalamus (VMH) of female, but not male rats. Therefore, we examined the effect of gonadectomy with or without four weeks of hormone treatment on PE mRNA expression in adult male Fisher 344 rats using quantitative in situ hybridization histochemistry. Gonadectomy reduced plasma testosterone and dihydrotestosterone (DHT) levels compared to intact rats, while subsequent estradiol (E2) or DHT treatment increased plasma E2 and DHT levels, respectively. Furthermore, gonadectomy reduced PE mRNA expression in the VMH, but not in the striatum nor the olfactory tubercle compared to intact rats, and this reduction was prevented in the presence of E2, but not DHT. The results suggest that the activation of estrogen receptors normally functions to maintain the level of VMH PE mRNA expression, which is sensitive to hormonal regulation in adult male rats. Thus gonadal steroid hormones might regulate those reproductive functions which are modulated by PE-derived opioid peptides in the male rat brain.

Sensitization of neuronal response to cocaine during withdrawal following chronic treatment.

The neural basis of sensitization was examined by assessing the expression of zif/268 mRNA in cortical and striatal neurons following cocaine challenge (30 mg kg-1, i.p.) after 2 weeks of daily treatment with cocaine or saline vehicle. Acute cocaine challenge induced zif/268 mRNA expression in neurons of the caudate putamen (CP), nucleus accumbens (NAc) shell and prelimbic cortex. Chronic cocaine treatment attenuated zif/268 mRNA expression in these regions, although zif/268 mRNA response in CP neurons recovered after 7 days of withdrawal. In contrast, cocaine challenge during withdrawal augmented zif/268 response in neurons of the infralimbic cortex and olfactory tubercle. Sensitized activity in these regions could serve to amplify mesolimbic dopamine and NAc responses, and might provide a critical stimulus to drug craving.

Social defeat stress increases expression of mu-opioid receptor mRNA in rat ventral tegmental area.

Prompted by previous studies linking social defeat stress to changes in opioid antinociception, we evaluated the expression of mu-opioid receptor (MOR)-encoding mRNA in selected rat brain areas as a function of this type of stress. Because opioids mediate significant regulatory activities of brain dopamine neurons, dopaminergic loci in the ventral tegmental area (VTA) and substantia nigra (SN) were selected for analysis. Within 30 min after social defeat stress, the level of MOR-encoding mRNA, as detected and quantified by in situ hybridization histochemistry, increased in the lateral VTA and this increase was present for at least 6 h. In contrast, defeat stress was without effect on the expression of MOR-encoding mRNA in the SN. These data suggest that stress-induced alteration of MOR-encoding mRNA expression in the VTA may be involved in the consequences of social defeat stress.

Neural mechanisms of tolerance to the effects of cocaine.

Chronic use of cocaine in high doses can produce tolerance as assessed by various behavioral, neurochemical, cellular and molecular measures in specific brain regions. Tolerance to cocaine is indicated by drug discrimination and intracranial self-stimulation models, which show the development of tolerance after approximately 1 week of frequent cocaine treatment, with recovery after a similar period of cocaine abstinence. Tolerance to the reinforcing properties of cocaine depends on dose, duration and frequency of cocaine self-administered by experimental animal or human subjects. The mechanism underlying this effect may involve an absolute or relative attenuation of dopamine response to cocaine challenge after frequent or repeated treatment in the nucleus accumbens (NAc). Similarly, afferent and efferent NAc circuits exhibit reduced metabolic activity, which lasts throughout the early period of withdrawal following repeated treatment. Attenuation of immediate early gene response also occurs, which might be related to a functional desensitization of dopamine D1-like receptors. Furthermore, intracellular adaptive responses to chronic cocaine exposure induce striatal dynorphin expression decreasing the behavioral potency of subsequent drug treatment. Thus, a combination of various pharmacodynamic mechanisms and the attenuation of dopamine response induced by sufficient dose, duration and frequency of cocaine exposure ultimately invoke the transient development of tolerance to the reinforcing effects of cocaine.

The influence of pre- and postnatal undernutrition on the developing brain stem reticular core: a quantitative Golgi study.

Neurons of the brain stem reticular core were examined in rats which had been exposed to prenatal or postnatal undernutrition. Prenatal undernutrition was induced by restricting maternal diet to 2/3 of ad libitum prior to and during the gestation period. Postnatal restriction to 20 days of age was induced by increasing the litter to 14 and removing the dam for 8 h each day. Both imposed experimental conditions tended to delay the course of reticular neuron development as indicated by altered number of dendritic protospines and varicosities. However, the effects of prenatal undernutrition were more severe. At birth, the amount of DNA in the brain stem was reduced (-24%) while average dendritic extent and branching were greatly increased. Early undernutrition appears to have a profound effect on the quality of the neuropil, both neuronal and glial, while postnatal exposure does not influence the structure of the dendritic arbor.

The sexually dimorphic region of the preoptic area in rats contains denser opiate receptor binding sites in females.

Quantitative autoradiographic analysis of opiate receptor binding using [3H]naloxone shows higher levels in the sexually dimorphic region of the medial preoptic area in female rats than in males. Opiate receptor density varies across the estrous cycle being densest in diestrous females. The sexually dimorphic nucleus of the preoptic area lies within the opiate receptor-rich region. Endogenous opiates in the medial preoptic region acting at opiate receptors which are of differential density in males and females could influence sex-specific behavior mediated by the region.

Mu-opiate receptor binding in the medial preoptic area is cyclical and sexually dimorphic.

The density and distribution of mu- and kappa-opiate receptors in the medial preoptic area (MPOA) of male and female rats across the estrous cycle was examined using quantitative in vitro autoradiography of [3H]D-Ala2,MePhe4,Gly-ol5-enkephalin (DAGO), [3H]naloxone and [3H]bremazocine binding. While no difference in kappa-receptor labeling was observed across sex or estrous stage, selective mu-receptor labeling with [3H]DAGO revealed a significant variation of density and distribution in the MPOA across the estrous cycle and between sexes. A dense concentration of mu-receptors located in the central, sexually dimorphic portion of the MPOA was observed during metestrus and diestrus in females, but not during proestrus nor in males. This region appeared to be the same as that labeled similarly using [3H]naloxone. These results suggest that a regional substrate for functional activation by endogenous opioid peptides (e.g. beta-endorphin) is cyclically regulated in females, which may explain the gonadal steroid-dependent effects of MPOA beta-endorphin on lordosis and luteinizing hormone secretion in females.

The sex hormone-dependent development of opiate receptors in the rat medial preoptic area.

The opiate receptor content of the sexually dimorphic medial preoptic area (MPOA) was examined in newborn and 5-day old (D6) male and female rats. A significant increase of [3H]naloxone binding was observed in and around the sexually dimorphic nucleus of the preoptic area (SDN-POA) in D6 female rats, relative to newborn females. Opiate receptor labeling did not increase over this period in males, nor was labeling different between males and females at birth. This dramatic alteration of MPOA opiate receptor content was observed to occur in either sex in the absence of testosterone postnatally; that is, neonatally-castrated males exhibited the same increase of labeling by D6 as did normal females. Conversely, daily postnatal testosterone treatment of females from birth to D6 resulted in the development of male-like MPOA opiate receptor pattern. The sex hormone-dependence of MPOA opiate receptor development is discussed in relation to the sex hormone-dependent ontogeny of SDN-POA structure. The overlap of critical periods for the development of these structural and chemical sexual dimorphisms suggests a role for endogenous opioids in modulating MPOA development.

Preoptic area opioids and opiate receptors increase during pregnancy and decrease during lactation.

Opiate receptor and endogenous opioid content were determined in pregnant, lactating, ovariectomized, and ovariectomized and subsequently estradiol- and progesterone-treated adult female rats. Levels of estradiol and progesterone produced by Silastic capsules implanted in animals of the ovariectomized, hormone-treated group were similar to natural levels of those hormones induced during pregnancy. Quantitative receptor autoradiography and radioimmunoassay were used to determine [3H]naloxone binding density and immunoreactive beta-endorphin content, respectively, in the preoptic area of the hypothalamus. Both opiate receptor binding density and beta-endorphin content in the preoptic area varied in the same direction in all experimental groups. The highest levels of both were observed during pregnancy and the lowest levels during lactation. Ovariectomy without subsequent hormone treatment produced intermediate levels of both opiate receptor and beta-endorphin. Ovariectomy with experimentally-induced estradiol and progesterone levels similar to those of pregnancy produced opiate receptor density and beta-endorphin content similar to those observed in pregnant animals. These data suggest that gonadal steroids are capable of altering function of the endogenous opiate system in the preoptic area. Moreover, preoptic area levels of opioids and opiate receptors are normally elevated during pregnancy and reduced during lactation. Since opiates are known to disrupt ongoing maternal behavior, a reduction of preoptic opiate function during lactation may be required to promote normal maternal behavior. The specific preoptic region involved in opiate regulation of maternal behavior may be illustrated by the zone of opiate receptor alteration observed herein.