Dynamics of noradrenergic circadian input to the chicken pineal gland

To analyze the dynamics of sympathetic input to the chicken pineal the concentrations of catecholamines, indoleamines and some of their metabolites were determined by high performance liquid chromatography with electrochemical detection (HPLC-EC) in the pineal glands of young chickens killed at different times of day. Rhythmic variations over 24 h were observed in tissue levels of dopamine (DA), 5-hydroxytryptamine (5-HT), N-acetylserotonin (NAS) and 5-hydroxyindoleacetic acid (5-HIAA), while norepinephrine (NE) concentrations exhibited no significant change. DA content peaked 2 h after onset of darkness and NAS was detectable only during the night. A bimodal pattern of 5-HT and 5-HIAA levels was observed with peak tissue levels occurring at dawn and dusk. To determine the possible differential effects of light on these biogenic amines, birds were sacrificed at midday, midnight and at midnight following a 1 h exposure to light, and their pineals processed for HPLC-EC. NE, DA and 5-HT levels were similar at midday and midnight, while 5-HIAA and NAS were elevated during the night. Midnight illumination decreased NE and NAS levels, increased 5-HT and 5-HIAA levels and had no effect on DA levels. Temporal variations in NE turnover were determined by pretreating young chickens with alpha-methyl-p-tyrosine, a tyrosine hydroxylase inhibitor, and measuring the rates of decline in NE content over 2 h at midday and midnight in birds held on light cycles and at mid-subjective day in birds held in constant darkness (DD).(ABSTRACT TRUNCATED AT 250 WORDS)     

Origins of the sympathetic projections to rat thyroid and parathyroid glands

The present study aimed to characterize the localization and pathways of sympathetic neurons innervating the thyroid and parathyroid glands. In rats subjected to unilateral superior cervical ganglionectomy or unilateral decentralization of the superior cervical ganglion 7 days earlier, ipsilateral depression of thyroid norepinephrine (NE) and epinephrine content to 6-16% of the contralateral intact lobe was observed. In both groups of animals neuronal [3H]NE uptake by the ipsilateral thyroid lobe was suppressed. In unilaterally decentralized rats pineal catecholamine levels remained within normal values whereas in unilaterally ganglionectomized rats a 74% decrease of pineal NE and E content was found. Unilateral section of the external carotid nerve abolished, as did unilateral superior cervical ganglionectomy, neuronal [3H]NE uptake in the ipsilateral thyroid lobe. In contrast external carotid nerve section did not modify the neuronal uptake of [3H]NE in the pineal gland. Either unilateral superior cervical ganglionectomy or external carotid nerve section resulted in similar involution of ipsilateral thyroid lobes of hypophysectomized rats. These results indicate that postganglionic sympathetic perikarya innervating the thyroid-parathyroid territory are located in the middle and/or inferior cervical ganglia and send their axons through the SCG and the external carotid nerve to these glands.     

Diurnal variation in norepinephrine-stimulated release of pineal serotonin in vitro

Summary Adult, male rats were maintained under 12L12D with lights on at 06.00 h. Their pineal glands were incubated at 37C in the presence or absence of 10^–4 M norepinephrine (NE). 5-HT and various metabolites were quantitated in post-incubation media and pineal glands by high performance liquid chromatography coupled with electrochemical detection. No differences were observed in the quantities of 5-HT released by pineal glands in four hour incubations starting at either 06.00, 13.00 or 18.00 h; however, a highly significant decrease below these levels was observed at 01.00 h. NE significantly stimulated 5-HT release at 13.00 and 18.00 h, but was ineffective at 01.00 and 06.00 h. These results confirm recently reported stimulatory effects of NE on the release of 5-HT into pineal gland incubation medium and further suggest a diurnal rhythm of pineal gland sensitivity to NE in vitro with maximum stimulation of 5-HT release at midphotophase.     

Sympathetic regulation of chicken pineal rhythms

Adult hens were chronically cannulated and held in light-dark (LD) 12:12 h lighthing regimes or in constant darkness (DD). Periodic blood sampling for 5–9 days revealed circadian rhythms in plasma melatonin titres. Superior cervical ganglionectomy (SCG-X) performed 1 week after hatching had little or no effect on these rhythms in LD, but unlike normals. SCG-X birds did not sustain persistent rhythms in DD. In SCG-X birds, norepinephrine (NE) infusion for 12 h of each 24 h in DD significantly reduced plasma melatonin titres during the infusion and re-established a rhythm.After each experiment, hens were killed, their pineals were removed and assayed by HPLC-EC for NE. dopamine (DA). serotonin (5-HT) and 5-hydroxy-3-indole-acetic acid (5-HIAA). SCG-X resulted in a 90% depletion of pineal NE: DA content was reduced to undetectable levels. Pineal 5-HT and 5-HIAA were also reduced by SCG-X.The chicken pineal contains circadian oscillators which persist in vitro^8.19.29. The results reported here suggest that noradrenergic fibres from the SCG regulate the pineal's inherent rhythmicity. NE normally released from sympathetic terminals during the bird's day may synchronize oscillators within the pineal by inhibiting melatonin synthesis.     

Peripheral distribution of free dopamine and its metabolites in the rat

Summary Free dopamine (DA) and its metabolites, homovanillic acid (HVA) and 3–4 dihydroxyphenylacetic acid (DOPAC), have been measured and compared with norepinephrine (NE) concentrations in rat peripheral tissues using high performance liquid chromatography with electrochemical detection (HPLC-ED). Detectable amounts of DA and its metabolites were found in all the analyzed tissues. The highest levels were found in carotid body, sympathetic ganglia, urogenital tract and heart, the lowest in liver and lung. DA and DOPAC distribution was heterogeneous and unrelated to NE concentration. Both the variable value of the DA/DA+NE ratio and the presence of DA metabolites in peripheral tissues indicate that a portion of DA may be stored outside noradrenergic neurons and directly catabolized in specific DA pools.     

Radioimmunoassay for mouse nerve growth factor (NGF). Effects of thyroxine administration on tissue NGF levels

A sensitive and specific radioimmunoassay (RIA) has been developed for β-nerve growth factor (NGF) purified from the submaxillary gland of the adult male mouse. The RIA is sensitive to 13 pg/assay tube with an intra-assay coefficient of variation of 7% and an interassay coefficient of variation of 11.8%. Of thesubstances tested for immunological cross-reactivity in the RIA, only mouse epidermal growth factor caused significant displacement of the radiolabeled ligand; 11% at a dose of 10 μg/ml. Mouse tissue extracts diluted out parallel to the standard curve. The effects of altered thyroid function on tissue NGF concentration in adult male mice were studied using the RIA. Thyroxine administration (25 μg T4 i.p. daily for 12 days) caused significant increase in submaxillary gland and liver NGF content and concentration (P<0.001) compared to controls but had no significant effect on heart and kidney NGF levels. Propylthiouracil treatment (0.05% PTU in the drinking water for 21 days) led to a marked increase in thyroid gland weight (550% of control) but had no significant effect on NGF concentration in any of the organs studied with the exception of kidney where a 75% reduction in NGF concentration was observed. These studies indicate that thyroid hormones increase NGF content and concentration in the submaxillary gland and liver of adult male mice and are suggestive of increased NGF synthesis. Thyroid hormones may therefore play an important regulatory role in NGF metabolism in peripheral tissues of the adult mouse.     

Novel stressors affected catecholamine stores in socially isolated normotensive and spontaneously hypertensive rats

Catecholamines in some central (hypothalamus and hippocampus) and peripheral tissues (adrenal glands and heart auricles) of long-term socially isolated normotensive and spontaneously hypertensive rats exposed to novel immobilization stress were determined by a simultaneous single isotope radioenzymatic assay. Long-term isolation (21 days) produced depletion of hypothalamic norepinephrine (NE) stores and hippocampal dopamine (DA) stores in both normotensive and spontaneously hypertensive rats. Acute immobilization stress (2 h) significantly decreased NE and DA stores in hypothalamus and hippocampus of naive normotensive and spontaneously hypertensive rats controls. However, novel immobilization stress applied to normotensive rats previously subjected to long-term isolation produced no changes in catecholamine levels in hypothalamus, while resulting in somewhat higher depletion of NE stores in hypothalamus of spontaneously hypertensive rats treated in the same way. Novel immobilization stress decreased NE and DA stores in hippocampus of normotensive but was without effect on NE and DA stores of spontaneously hypertensive rats. Social isolation did not affect catecholamine stores in peripheral tissues but novel immobilization stress produced a significant decrease in catecholamine content. The results suggest that some central and peripherals tissues of spontaneously hypertensive rats and normotensive rats differ with regard to catecholamine content and that there are certain differences in their responsiveness to stress.     

Afferent regulation of neurotransmitter metabolism in perikarya and terminals of developing sympathetic neurons

Steady-state levels and turnover of the neurotransmitter, norepinephrine (NE), were measured in sympathetic perikarya and in two sympathetic target organs in the rat at various times during postnatal development. NE content in sympathetic perikarya in the superior cervical ganglion (SCG) increases 15-fold from birth to reach adult levels by 60 days. This increase in NE content parallels the increase in total protein in the ganglion. The rate of turnover of NE in the sympathetic perikarya increases slightly from birth to adulthood. Since the perikarya in the SCG project to a variety of different targets in the head and neck, NE metabolism was also examined in two terminal sympathetic plexuses, in the iris and in the submandibular gland (SMG). The terminal noradrenergic plexuses within these target organs do not mature with the same time course. In the iris, levels of NE increase 24-fold from birth until 90 days postnatally. Turnover of NE in sympathetic terminals in the iris at the time of birth is equivalent to that in the adult. In contrast, both the content and turnover of NE in sympathetic terminals in the SMG are very low at birth, and increase dramatically in the first month postnatally. Deafferentation of the SCG at birth impairs the development of normal levels of NE in sympathetic perikarya by approximately 40%, and total ganglionic protein is similarly affected. NE turnover in sympathetic perikarya deafferented at birth is only slight reduced from normal. The response to neonatal deafferentation differs in the two terminal sympathetic plexuses. In neurons that project to the iris, no detectable NE turnover could be measured, although the content of transmitter attains 64% of control values after deafferentation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulatory effects of melatonin on behavior, hemolymph metabolites, and neurotransmitter release in crayfish

Melatonin affects a variety of circadian processes such as behavior and neurotransmitter release in vertebrates. Crayfish melatonin production occurs in the eyestalks, and the cycle of production may change seasonally. To date, however, melatonin's roles and mechanisms of action in crustacean physiology are unclear. We injected melatonin or saline into crayfish in scotophase and monitored activity and hemolymph glucose/lactate over 24 h in early spring. Crayfish were significantly more active in photophase versus the expected scotophase, and had concurrent glucose/lactate peaks. Melatonin reversed the activity pattern, causing a scotophase activity peak, but not the glucose/lactate patterns. This study was repeated in late summer, during which control activity and glucose/lactate levels were elevated in scotophase. Melatonin decreased the amplitude of scotophase activity and glucose/lactate, eliminating activity and glucose cycles. We also injected melatonin or saline at various times of day in early summer and monitored locomotor activity for 1 h. Controls had high activity at 1200 (mid-photophase) and 2100 h (early scotophase), and melatonin increased activity at 1200 h but decreased it at 2100 h. Melatonin also increased activity at 1500 h but not 1800 h (late photophase). Next, we examined the influence of melatonin on crayfish neurophysiology. Melatonin (10 microM) enhanced synaptic transmission at the neuromuscular junction (NMJ). The presynaptic action resulted in more vesicles being released during evoked stimulation. Our study indicates that melatonin may have a phylogenetically conserved role in the transduction of circadian information in invertebrates as in vertebrates. Behavioral and physiological effects may be mediated by modulation of central pathways, enhanced at the peripheral level via neuromodulation of the NMJ.     

Gzα deficient mice: enzyme levels in the autonomic nervous system, neuronal survival and effect of genetic background

Our laboratory has generated a genetically mutant mouse in which the alpha subunit of the heterotrimeric GTP binding protein, G(z) has been made dysfunctional by homologous recombination to determine its in vivo function. These animals show a characteristic failure to thrive phenotype. G(z alpha) is expressed in a variety of nervous system tissues as well as in the adrenal medulla. We therefore examined the autonomic nervous system of the G(z alpha) deficient mouse by measuring the activity of tyrosine hydroxylase and choline acetyltransferase in the superior cervical ganglia, submaxillary gland and the adrenal medulla. Preliminary results using animals of mixed BALB/c and C57BL/6 strains gave inconsistent results. Further experiments demonstrated differences in the activity of tyrosine hydroxylase and choline acetyltransferase between BALB/c and C57BL/6 mouse strains. The analysis of the pure strains showed a reduction in the size and enzyme levels of the adrenal gland and submaxillary glands of the G(z alpha) deficient mouse suggesting a role for adrenal insufficiency and/or nutritional disorders for the failure to thrive phenotype. The survival of sympathetic and sensory neurons was also examined in the G(z alpha) deficient mouse and in the presence of pertussis toxin, sympathetic but not sensory neuronal survival in G(z alpha) deficient mice was significantly attenuated. This suggests that in vivo other pertussis toxin sensitive G proteins may be recruited to compensate for the loss of G(z alpha).     

Differential effect of guanethidine on dopamine and norepinephrine in rat peripheral tissues

Summary The changes of dopamine (DA) and norepinephrine (NE) were investigated in rat peripheral tissues after guanethidine treatment (50 mg/kg i.p. five days each week) during one week (group 1, n=10, five injections) and during 2.5 weeks (group 2, n=8, 13 injections).Guanethidine greatly reduced NE levels in all the analyzed tissues but only partially depleted DA in kidney, bladder, stomach, intestine, lung and liver and in sympathetic ganglia.The differential pattern of changes between DA and NE induced by guanethidine suggests that peripheral DA is distributed in several neuronal or non-neuronal pools, whose presence, nature and contribution varies in the different tissues. Both noradrenergic cell bodies and small intensely fluorescent cells (SIF cells) can contribute to the DA in the superior cervical ganglion.Noradrenergic neurons seem to be the main sources of DA in seminal vesicles, vas deferens, heart and spleen.In addition to noradrenergic nerves, extraneuronal sources could account for a meaningful portion of DA in kidney, gastrointestinal tract, lung and liver. The bladder is the peripheral tissue where DA exhibits the highest resistance to the neurotoxin. Accordingly, these tissues may provide meaningful sources of non-precursor DA pools.     

Autoradiographic localization of peripheral M1 muscarinic receptors using [H]pirenzepine

A novel antimuscarinic agent, pirenzepine, has been proposed to distinguish at least two subtypes of muscarinic receptor. M1 receptors have been designated as those displaying a high affinity for pirenzepine. Both functional and binding studies have revealed a prevalence of M1 receptors in sympathetic ganglia while autonomic effector tissues have only low densities of M1 receptors. In the present study, in vitro autoradiographic procedures have been used to localize specifically high affinity binding sites for pirenzepine (M1 receptors) in sections of guinea-pig ileum, rat superior cervical ganglion and rat submaxillary gland. The overall localization of muscarinic receptors was also studied using the non-selective antagonist, [³H]N-methylscopolamine. The highest densities of M1 receptors were found in superior cervical ganglion, sympathetic nerve bundles, myenteric ganglia and mucous secreting cells of the submaxillary gland, while lower densities were found in smooth muscle and serous secreting cells of the submaxillary gland. No area found to possess muscarinic receptors was devoid of M1 receptors.     

Autoradiographic localization of peripheral M1 muscarinic receptors using [3H]pirenzepine

A novel antimuscarinic agent, pirenzepine, has been proposed to distinguish at least two subtypes of muscarinic receptor. M1 receptors have been designated as those displaying a high affinity for pirenzepine. Both functional and binding studies have revealed a prevalence of M1 receptors in sympathetic ganglia while autonomic effector tissues have only low densities of M1 receptors. In the present study, in vitro autoradiographic procedures have been used to localize specifically high affinity binding sites for pirenzepine (M1 receptors) in sections of guinea-pig ileum, rat superior cervical ganglion and rat submaxillary gland. The overall localization of muscarinic receptors was also studied using the non-selective antagonist, [3H]N-methylscopolamine. The highest densities of M1 receptors were found in superior cervical ganglion, sympathetic nerve bundles, myenteric ganglia and mucous secreting cells of the submaxillary gland, while lower densities were found in smooth muscle and serous secreting cells of the submaxillary gland. No area found to possess muscarinic receptors was devoid of M1 receptors.     

Alterations in monoamines level in discrete brain regions and other peripheral tissues in young and adult male rats during experimental hyperthyroidism

The present study was conducted to investigate the effect of experimentally-induced hyperthyroidism on dopamine (DA), norepinephrine (NE) and serotonin (5-HT) levels in different brain regions as well as in blood plasma, cardiac muscle and adrenal gland of young and adult male albino rats (60 rats of each age). Hyperthyroidism was induced by daily s.c. injection of L-thyroxine (L-T₄, 500 μg/kg body wt.) for 21 consecutive days. Induction of hyperthyroidism caused a significant elevation in DA and 5-HT levels in most of the tissues studied of both young and adult animals after 7, 14, and 21 days. NE content significantly decreased after 21 days in most of the brain regions examined and after 14 and 21 days in blood plasma of young rats following hyperthyroidism. In adult rats, NE content decreased after 14 and 21 days in cardiac muscle and after 21 days only in adrenal gland. It may be suggested that the changes in monoamines level induced by hyperthyroidism may be due to disturbance in the synthesis, turnover and release of these amines through the neurons impairment or may attributed to an alteration pattern of their synthesis and/or degradative enzymes or changes in the sensitivity of their receptors     

Circadian profiles of focal epileptic seizures: a need for reappraisal

Circadian rhythm of seizure is underestimated in the study of focal epilepsies. A review of the current literature revealed a clear correlation between cortical epileptogenic focus and the circadian phase of seizure peak occurrence in adult patients. A single diurnal peak at 19:00 was found in seizures originating from the occipital lobe, between 5:00 and 7:00 in frontal lobe seizures, and between 16:00 and 17:00 h in temporal lobe seizures. Two diurnal peaks, between 5:00 and 7:00, and at 23:00 are reported in seizures from the parietal lobe, and between 7:00 to 8:00 and 16:00 to 17:00 in mesial temporal onset seizures. This circadian character of seizure occurrence in focal epilepsies may not be unique to partial seizures since recent clinical and experimental data indicate that generalized seizures also demonstrate circadian effects. The clinical evidence on generalized seizures and epilepsies is not recent, but a formal integration of circadian rhythmicity in our understanding and clinical management of epilepsies may be warranted.     

Differential effect of interferon-gamma injection on mitogenic responses in sympathetically denervated submaxillary lymph nodes of rats

This study aimed to assess the effect of interferon (IFN)-gamma treatment on mitogenic responses in submaxillary lymph nodes in the presence or absence of local sympathetic nerves. Adult male rats were subjected to a unilateral superior cervical ganglionectomy and to a contralateral sham-operation. Seven days later, rats received five i.p. daily injections of human IFN-gamma (10(5) U.I./kg) or saline. On the day after the last injection, rats were killed at six different times throughout a 24-h cycle and the mitogenic effect of lipopolysaccharide (LPS) and concanavalin A (Con A) was assessed in single-cell suspensions of lymph nodes. In vehicle-treated rats, proliferation responses to LPS in innervated lymph nodes did not show time-of-day variations while those in denervated lymph nodes attained a maximum at 17:00 h. Following IFN-gamma administration, a promoting effect of LPS mitotic response was detected at 01:00 h at the innervated side only. As far as the mitogenic responses to Con A, proliferation in innervated lymph nodes of vehicle-treated controls attained a maximum at 09:00 h. Such a daily variation in response to Con A was not detectable at the denervated side. IFN-gamma treatment increased significantly Con A activity by promoting a greater mitogenic response at 01:00 h. Sympathetic denervation of lymph nodes brought about a shift in the maximum in number of cells per mg of lymph node from 21:00 to 13:00 h. After IFN-gamma treatment, maxima in cell number occurred at 05:00 h at both the innervated and denervated side. The results indicate that IFN-gamma effects in rat submaxillary lymph nodes are under substantial modulation by local sympathetic nerves.     

Substance P in the superior cervical ganglion and the submaxillary gland of the rat

Substance P has been detected in the superior cervical ganglion and submaxillary gland of the rat by radioimmunoassay. Decentralization, section of the carotid sinus nerve, or deefferentation does not affect the level of substance P in the rat superior cervical ganglion. Superior cervical ganglionectomy does not affect the amount of substance P in the submaxillary gland; however, section of the chorda tympani or duct ligation significantly reduces substance P in the submaxillary gland. It is concluded that substance P in the superior cervical ganglion is not located in the preganglionic sympathetic neurons, afferents from the glossopharyngeal nerve or post-ganglionic neurons projecting to the submaxillary gland or carotid body. Substance P does not appear to be associated with sympathetic innervation of the submaxillary gland, but it is anatomically and functionally related to the chorda tympani and possibly involved in the parasympathetic innervation of the gland. Substance P may also be present in gland cells of the submaxillary gland.     

Conjugated HVA increase in rat urine after insulin-induced hypoglycemia: Involvement of central dopaminergic structures but not of adrenal medulla

Summary To examine the possible contribution of Rat adrenal medulla to urinary DOPAC and HVA, we have studied these compounds in adrenals and urine under insulin-induced (5 IU/kg) hypoglycemic stimulation.In the urine samples collected over 16-hour-period following the intravenous insulin injection, there was a great increase in E, NE and their methoxylated derivatives MN and NMN, without change in DA, DOPAC, MHPG and free HVA excretion. In addition, there was a pronounced increase in urinary HVA conjugates (glucuronide and sulfate). Only very low amounts of DOPAC (10±2ng/gland; 0.05% of catechols) and no detectable amounts of HVA (<3 ng/gland) were found in adrenal glands, without no significant change two hours after insulin, thus suggesting that Rat adrenal glands are not meaningful sources for urinary HVA and DOPAC.Since free HVA and total MHPG excretion remained unchanged, HVA contribution from sympathetic neurons seems unlikely in our study.In contrast, highly increased levels of conjugated HVA-and at a lesser extent of conjugated DOPAC-have been found in the striatum, which appears to be the most likely source of urinary HVA increment. The dopaminergic activation following insulin affected too the hypothalamus but not the nucleus accumbens. The role of such central dopaminergic activation has been discussed in terms of feeding behavior.     

Toxin-induced inhibition of nerve terminal growth

An in vivo model to permit the quantitative study of the effects of neurotoxins upon nerve terminal growth has been developed and used in practice to test the ability of methyl mercury chloride, leptophos, ethanol, morphine and acrylamide to inhibit nerve terminal growth. The model utilized the sympathetic innervation of rat submaxillary salivary glands. Nerve terminal growth was induced following crush of the sympathetic axons which innervated the submaxillary gland and quantified by measuring the return towards normal values of the density of sympathetic innervation in denervated glands. The nerve crush reduced the density of sympathetic innervation to a very low value but nerve growth began within 9 days and was sufficient to restore the density of innervation to approximately 70% of normal values in three weeks. Restoration of the original density of innervation took between 40 and 60 days. The major advantage of the model is its ability to detect and quantify 30% - 100% inhibition of nerve terminal growth. The major disadvantage is the time required to measure the density of sympathetic innervation though this can be minimized in a number of ways which are discussed. Acrylamide inhibited sympathetic nerve terminal growth without causing degeneration of sympathetic neurones. This demonstrates that toxins can selectively inhibit nerve terminal growth which will result in altered neuronal circuitry and abnormal function. Methyl mercury chloride, leptophos, ethanol and morphine did not inhibit sympathetic nerve terminal growth.     

Sex dimorphic alterations in postnatal brain catecholarnines after gestational morphine

The concentration of brain catecholamines was measured in the hypothalamus, preoptic area (POA), frontal cortex, cerebellum, and striatum of rats exposed in utero to morphine (5–10 mg/kg/twice daily) during gestation days 11–18. Prenatal morphine induced regionally specific, sexually dimorphic alterations in male and female norepinephrine (NE), and dopamine (DA) content at different postnatal ages. Prenatal morphine significantly increased NE content in the hypothalamus of both sexes at postnatal day (PND) 23. In the POA, on the other hand, morphine increased NE content in exposed males at PND 23 and in females at PND 33. In the cerebellum, the NE content of both sexes was significantly elevated at PND 45. In the striatum, NE content was increased by the prenatal morphine only in females at PND 16. The concentration of DA was also affected in a sexually dimorphic manner. At PND 16, prenatal morphine increased the levels of hypothalamic DA only in males, and it reduced the content of DA in female but not male PDA. At PND 45, prenatal morphine increased DA in the hypothalamus of females and decreased it in males. In the cerebellum of 16-day-old morphine-exposed animals, DA levels were increased only in males; at PND 45, the levels of DA were still increased in males but had not changed in females. In the striatum, the DA content was reduced only in males at PND 16. Thus, prenatal morphine alters the development of both NE and DA neurotransmitter systems in the hypothalamus, POA, striatum, and cerebellum in a sexually dimorphic manner.