Ontogeny of cholecystokinin-like immunoreactivity in the Brazilian opossum brain.

We have studied the anatomical distribution of cholecystokinin-like immunoreactive (CCK-IR) somata and fibers in the brain of the adult and developing Brazilian short-tailed opossum, Monodelphis domestica. Animals ranged in age from the day of birth (1PN) to young adulthood (180PN). A nickel enhanced, avidin-biotin, indirect immunohistochemical technique was used to identify CCK-IR structures. Somata containing CCK immunoreactivity were observed in the cerebral cortex, hippocampus, hypothalamus, thalamus, midbrain, and brainstem in the adult. Cholecystokinin immunoreactive fibers had a wide distribution in the adult Monodelphis brain. The only major region of the brain that did not contain CCK-IR fibers was the cerebellum. The earliest expression of CCK immunoreactivity was found in fibers in the dorsal brainstem of 5-day-old opossum pups. It is possible that the CCK-IR fibers in the brainstem at 5PN are of vagal origin. Cholecystokinin immunoreactive somata were observed in the brainstem on 10PN. The CCK-IR cell bodies observed in the brainstem at 10PN may mark the first expression of CCK-IR elements intrinsic to the brain. A broad spectrum of patterns of onset of CCK expression was observed in the opossum brain. The early occurrence and varied ontogenesis of CCK-IR structures indicates CCK may be involved in the function of a variety of circuits from the brainstem to the cerebral cortex. The early expression of CCK-IR structures in the dorsal brainstem suggests that CCK may modulate feeding behavior in the Monodelphis neonate. Cholecystokinin immunoreactivity in forebrain structures such as the suprachiasmatic nucleus, medial preoptic area, thalamus and cortical structures indicates that CCK may also be involved in circadian rhythmicity, reproductive functions, as well as the state of arousal of the Brazilian opossum. The ontogenic timing of CCK immunoreactivity in specific circuitry also indicates that CCK expression does not occur simultaneously throughout the brain. This pattern of CCK onset may relate to the temporal need for CCK in specific circuits of the central nervous system (CNS) during development.     

Galanin-like immunoreactivity in the adult and developing Brazilian opossum brain.

The distribution of galanin-like immunoreactivity has been characterized in the brain of the adult and developing Brazilian opossum (Monodelphis domestica). Two commercially available antisera were used to examine the distribution of galanin-like immunoreactive (GAL-IR) cells and fibers. Nuclear groups containing GAL-IR cell bodies and fibers were seen throughout the adult opossum brain. The distribution of GAL-IR elements seen is similar to that reported for other mammals. Based on these findings, we believe that galanin may have similar physiological functions in the adult Brazilian opossum as has been reported for other mammals. In the developing brain, GAL-IR structures were seen as early as 1 day postnatal (PN) in the developing hypothalamus and brainstem. By days 5 and 10 PN, there was a robust expression of galanin-like immunoreactivity in specific regions of the brain. Since neurogenesis and brain morphogenesis are actively occuring postnatally in the opossum, galanin may be playing a role in the differentation of specific regions of the brain.     

Ontogeny of cells containing estrogen receptor-like immunoreactivity in the Brazilian opossum brain.

In this study, we have used the Brazilian short-tailed opossum (Monodelphis domestica) as a model to study the ontogeny of estrogen receptors in the mammalian brain. Monodelphis is a small, pouchless marsupial which breeds well under laboratory conditions and whose young are born in an immature sexually undifferentiated state. The Abbott H222 monoclonal rat estrogen receptor antibody (gift of Abbott Laboratories) was utilized in an indirect immunohistochemical procedure to detect estrogen receptors in developing opossum brains. Estrogen receptors were first expressed in the dorsomedial and ventromedial hypothalamus of the opossum 10 days after birth (10PN). Most regions that contained estrogen receptor-like immunoreactivity (ER LI) in the adult opossum contained ER LI at 15 PN. These areas include the lateral septum, medial preoptic area, bed nucleus of the stria terminalis, periventricular preoptic area and hypothalamus, amygdala, dorsomedial and ventromedial hypothalamic nuclei, arcuate nucleus, ventral premammillary nucleus, and the midbrain central grey. The number of cells that contain ER LI increased through 60PN in all regions that will contain ER LI in the adult opossum. These results indicate that estrogen receptors are present in early development of the Monodelphis brain and may mark the beginning of a critical period for sexual differentiation of the opossum brain.     

Characterization and ontogeny of synapse-associated proteins in the developing facial and hypoglossal motor nuclei of the Brazilian opossum

The characterization and ontogeny of synapse-associated proteins in the developing facial and hypoglossal motor nuclei were examined in the Brazilian opossum (Monodelphis domestica). Immunohistochemical markers utilized in this study were the synaptic vesicle-associated proteins synaptophysin and synaptotagmin; a synaptic membrane protein, plasma membrane-associated protein of 25 kDa (SNAP-25); a growth cone protein, growth-associated phosphoprotein-43 (GAP-43); and the microtubule-associated proteins axonal marker τ and dendritic marker microtubule-associated protein-2 (MAP-2). In this study, we have found that during the first 10 postnatal days (1–10 PN), the facial motor nucleus lacked immunoreactivity for synaptophysin, synaptotagmin, GAP-43, τ, and SNAP-25. After 10 PN, immunoreactivity increased in the facial motor nucleus for synaptophysin, synaptotagmin, GAP-43, and τ, whereas immunoreactivity for SNAP-25 was not evident until between 15 and 25 PN. Conversely, immunoreactivity for MAP-2, was present in the facial motor nucleus from the day of birth. In contrast, the hypoglossal motor nucleus displayed immunoreactivity from 1 PN for synaptophysin, synaptotagmin, SNAP-25, GAP-43, τ, and MAP-2. These results suggest that the facial motor nucleus of the opossum may not receive afferent innervation as defined by classical synaptic markers until 15 PN and, further, that characteristic mature synapses are not present until between 15 and 25 PN. These results indicate that there may be a delay in synaptogenesis in the facial motor nucleus compared to synaptogenetic events in the hypoglossal motor nucleus. Because the facial motor nucleus is active prior to completion of synaptogenesis, we suggest that the facial motoneurons are regulated in a novel or distinct manner during this time period. © 1996 Wiley-Liss, Inc.     

Localization of cells containing estrogen receptor-like immunoreactivity in the Brazilian opossum brain

The Brazilian opossum (Monodelphis domestica) is a small, pouchless marsupial whose young are born in an immature, sexually undifferentiated state. Etgen and Fadem, and Handa and coworkers have biochemically detected and characterized estrogen receptors in the forebrain of the Brazilian opossum. In this study, we have examined the distribution of estrogen receptor-like immunoreactive (ER-LI) cells in the brains of gonadectomized male and female Brazilian opossums using Abbott H222 rat monoclonal estrogen receptor antibody (H222 is a gift of Abbott Labs). An indirect immunohistochemical procedure employing the Vectastain Elite system and a nickel-enhanced DAB chromogen was used. A large number of ER-LI cell nuclei were observed in the medial preoptic area, ventral septal nucleus, medial division of the bed nucleus of the stria terminalis, lateral part of the ventromedial hypothalamus, premammillary nucleus, arcuate nucleus, posterior amygdaloid nucleus, and the midbrain central grey. Lower numbers of ER-LI cell nuclei were observed in the intermediate subdivision of the lateral septal nucleus, and in the anterior, medial, and posterior cortical amygdaloid nuclei. The anatomical distribution of ER-LI in the Brazilian opossum brain is similar to that which has been reported for estrogen binding sites following biochemical analysis. Based on these findings, we believe specific regions of the Brazilian opossum brain may serve as substrata for the action of estrogen in the adult. In addition, these results are supportive of the use of this animal model to investigate the organizational effects of estrogen on the developing central nervous system.     

Early appearance and transient expression of vasopressin receptors in the brain of rat fetus and infant. An autoradiographical and electrophysiological study

The development of vasopressin (AVP) receptors in the rat brain, spinal cord and pituitary gland was studied by in vitro light microscopic autoradiography. AVP binding sites were labeled using [3H]AVP in tissue sections from animals aged between embryonic day 12 (E12) and postnatal day 90 (PN90); the binding of [3H]AVP to oxytocin receptors was prevented by adding in the incubation medium a highly selective oxytocin agonist. Specific binding was first detected at E16 in the ventral pontine reticular formation. Many other brain areas were progressively labeled between E18 and PN5. The distribution of binding sites observed at PN5 remained unchanged until the beginning of the third postnatal week. Thereafter binding was markedly reduced or even disappeared in several areas, in particular in the facial nucleus. The adult distribution of AVP binding sites was established at the time of weaning. The properties of transient AVP binding sites in the facial nucleus were studied both by autoradiography and by electrophysiology. Non-radioactive AVP displaced [3H]AVP binding in this nucleus as efficiently as it did in the lateral septum of the adult. Single-unit extracellular recordings showed that AVP can excite facial motoneurones by interacting with receptors which are pharmacologically indistinguishable from V1 (vasopressor) type. Thus, AVP binding sites transiently expressed in the brain of fetal and infant rat probably represent functional neuronal receptors, having the same ligand selectivity and affinity than AVP binding sites present in the adult. This suggests that AVP acts not only as a neuropeptide in the adult brain but may play a significant role during maturation of the central nervous system.     

Distribution and brainstem origin of cholecystokinin-like immunoreactivity in the opossum cerebellum

In order to determine the distribution of the peptide cholecystokinin (CCK) within the cerebellum and medullary precerebellar nuclei of the adult opossum, sections of these brain regions were processed for peroxidase-antiperoxidase immunohistochemistry. Within the inferior and superior cerebellar peduncles, fine-beaded fibers are evident and a beaded plexus of fibers is present in all the cerebellar nuclei. In the overlying cerebellar cortex, CCK-positive mossy fiber rosettes are present in all lobules, where their morphology varies from simple enlargements to more complex rosettes. However, their distribution varies particularly in vermal lobules II, III, VII, and IX where they are organized in parasagittal bands. Climbing fibers that are positive for CCK are present in very restricted areas of vermal lobules IV, VII, and VIII. After colchicine pretreatment, CCK-positive cell bodies are seen in restricted regions of the posterior interposed and fastigial nuclei as well as within several precerebellar nuclei known to give rise to mossy fibers. Such nuclei include the lateral cuneate nucleus, the nucleus prepositis hypoglossi, the nucleus reticularis lateralis, the nucleus raphe obscurus, the paramedian reticular nucleus, the nucleus reticularis gigantocellularis, and the medial vestibular nucleus. To localize the brainstem origin(s) of the CCK fibers in the cerebellum, a double-label paradigm employing a retrograde tracer and CCK immunohistochemistry was used. These experiments indicate that CCK mossy fibers originate primarily within the lateral cuneate nucleus, the perihypoglossal complex, and the lateral reticular nucleus. Some also originate within the medial vestibular nucleus and the nucleus reticularis gigantocellularis. In addition, double-labeled cell bodies are present within the caudal medial accessory inferior olive, the likely source of the CCK-positive climbing fibers. These data indicate that specific populations of climbing fibers and mossy fibers may utilize CCK to alter the firing rate of their target neurons.     

Localization of cholecystokinin binding sites in canine brain using quantitative autoradiography

1. CCK receptors have been characterized and localized in various mammalian species and significant species-specific differences in their distribution have been identified. In the present study, we report the first autoradiographic localization of CCK binding sites in the canine brain. 2. High densities of [125I]BH-CCK-8 binding sites were found in the cortex, cerebellum, hippocampus, caudate nucleus, olfactory bulb and nucleus accumbens. Moderate densities were present in the putamen, amygdala, and substantia gelatinosa. Low binding densities were observed in the globus pallidus, inferior colliculus, hypothalamus and thalamus. 3. Although the distribution profile of CCK binding sites in canine brain is similar to those previously reported in the rodent, primate and human brain, notable differences were observed in the hippocampus, cortex and cerebellum.     

Appearance and transient expression of oxytocin receptors in fetal, infant, and peripubertal rat brain studied by autoradiography and electrophysiology

The development of oxytocin (OT) receptors in the rat brain and spinal cord was studied by in vitro light microscopic autoradiography and by electrophysiology. OT receptors were labeled using a monoiodinated OT antagonist in tissue sections from animals aged between embryonic day 12 (E12) and postnatal day 90 (PN90); the response of ongoing spike activity to the addition of OT was assessed in neurons located in the dorsal motor nucleus of the vagus nerve of the neonate. Specific binding was detected first at E14 in a region that later differentiated into the dorsal motor nucleus of the vagus nerve. Many other regions were progressively labeled between E20 and PN5. From PN5 to PN16, the distribution of binding sites remained essentially unchanged but differed markedly from that characteristic of the adult. The change-over from the "infant pattern" to the "adult pattern" occurred in 2 stages: the first change took place between PN16 and PN22, a time corresponding to the preweaning period; the second change occurred after PN35 and thus coincided with the onset of puberty. During the first transition period, binding was reduced or disappeared in several areas intensely labeled at earlier stages, in particular, in the cingulate cortex and the dorsal hippocampus. At the same time, binding sites appeared in the ventral hippocampus. At puberty, high densities of OT binding sites appeared in the ventromedial hypothalamic nucleus and the olfactory tubercle. Electrophysiological activity was recorded from vagal neurons in slices obtained from animals sacrificed at PN1-PN12. OT and a selective OT agonist reversibly increased the firing rate of these neurons in a concentration-dependent manner. The neuronal responsiveness was similar to that reported previously in the adult. These results suggest that OT binding sites detected by autoradiography in the developing rat brain represent, at least in some areas, functional neuronal receptors.     

Autoradiographic localization of tryptamine binding sites in the rat and dog central nervous system

Tryptamine, an endogenous trace amine, is currently postulated to be a neuromodulator or neurotransmitter in the mammalian CNS. High-affinity binding sites have been described for tryptamine in rat brain homogenate preparations. The present study further characterizes tryptamine binding throughout the CNS and delineates its distribution using in vitro receptor binding in conjunction with autoradiographic techniques. Saturation studies on 20-micron-thick brain sections suggest a single class of binding sites (Hill coefficient = 0.97 +/- 0.04) with a high affinity (KD = 4.79 +/- 1.55 nM). In competition studies, kynuramine and tetrahydrobetacarboline significantly inhibited H3-tryptamine binding while serotonin, dopamine, and phenylethylamine failed to significantly inhibit it. The most potent inhibitor of H3-tryptamine binding was tryptamine (KI = 4.19 +/- 2.13 nM). In rat brain sections processed for in vitro autoradiography, highest binding occurred in the following limbic structures: the accumbens nucleus, the amygdalohippocampal area, the lateral septal nucleus, the entorhinal cortex, and the anterior olfactory nucleus. At diencephalic levels, the highest binding was observed in the reuniens thalamic nucleus, the paraventricular thalamic nucleus, the medial habenular nucleus, the central medial thalamic nucleus, and the arcuate hypothalamic nucleus. In the midbrain of the rat, binding was most notable in the interpeduncular nucleus, the superficial layer of the superior colliculus, the periaqueductal gray, and the paranigral nucleus. In the lower brain stem of the dog, binding was evident in the external cuneate nucleus, the spinal trigeminal nucleus, and in the region of the solitary nucleus. Binding was also present in both the ventral and dorsal horns of the canine spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and distribution of estrogen receptors in the diencephalon of the gray short-tailed opossum

The Brazilian gray short-tailed opossum is a pouchless marsupial whose young are born sexually undifferentiated making this animal ideal for developmental studies. Previously, Etgen and Fadem (Dev. Brain Res., 49 (1989) 131-133; Gen. Comp. Endocrinol., 66 (1987) 441-446) detected estrogen receptor (ER) in the hypothalamus-preoptic area, and compared males and females in the adult and during development. In this study we characterized the ER and determined its distribution in specific diencephalic regions in the brains of adult male and female opossums. ER were measured by the in vitro binding of [3H]estradiol to cytosol of microdissected brain nuclear regions. Radioinert moxestrol (R2858) was used to define non-specific binding. Saturation analysis showed a single high-affinity binding site. Binding was displaced by estradiol (E2), diethylstilbestrol (DES) and R2858, but not by non-estrogenic steroids. Ligand bound receptor adhered to DNA-cellulose and was eluted as a single peak with 0.2-0.3 M NaCl. High levels of ER were found in the medial preoptic-periventricular area. Intermediate levels were seen in the ventromedial hypothalamic nucleus, medial amygdala and arcuate nucleus. No sex differences were observed. The presence of a neural ER and its similarity of distribution to that of the laboratory rat support the use of this animal model in studies examining steroid dependent organization of the hypothalamus.     

Postnatal development of cholecystokinin (CCK) binding sites in the rat forebrain and midbrain: an autoradiographic study

The postnatal development of cholecystokinin (CCK) binding sites in the rat forebrain and midbrain was studied by in vitro receptor autoradiography. In the majority of structures, the densities of sites were low over the first week after birth, increased until the third week, and decreased over the fourth week to reach adult levels. However, both the rate of increase and the extent of the decrease varied in large proportions among structures. For instance, labeling in the neocortex underwent its largest increase from postnatal day 10, while this increase was already begun at day 7 in the paleocortex. On the other hand, over the fourth postnatal week, the densities could either remain roughly constant (cingulate cortex), slightly decrease (thalamic reticular nucleus), or even return to background levels (pyramidal layer of hippocampus). These different timetables may depend mostly on the differential growth of cells expressing the CCK receptor gene within the developing CNS. The absence of CCK binding sites in most of the regions during the early postnatal period precludes a major role of this peptide in the embryonic development of the rat brain. However, in some regions as the ventromedial hypothalamic nucleus, the endopyriform cortex or the medial nucleus of amygdala, 30-50% of the adult levels were already present at birth. Whether this observation reflects an earlier functional maturation of these structures or a direct participation of the corresponding CCK systems in their development remains to be established.     

Transplantation of neural progenitor cells into the developing retina of the Brazilian opossum: an in vivo system for studying stem/progenitor cell plasticity

In developing cell transplant strategies to repair the diseased or injured retina is essential to consider host-graft interactions and how they may influence the outcome of the transplants. In the present study we evaluated the influence of the host microenvironment upon neural progenitor cells (NPCs) transplanted into the developing and mature retina of the Brazilian opossum, Monodelphis domestica. Monodelphis pups are born in an extremely immature state and the neonatal pups provide a fetal-like environment in which to study the interactions between host tissues and transplanted NPCs. Three different populations of GFP-expressing NPCs were transplanted by intraocular injection in hosts ranging in age from 5 days postnatal to adult. Extensive survival, differentiation and morphological integration of NPCs were observed within the developing retina. These results suggest that the age of the host environment can strongly influence NPC differentiation and integration.     

Differential development of insulin-like growth factor-I binding in the hypothalamus of hamster and rat.

We investigated the binding of [125]insulin-like growth factor-I ([125I]IGF-I) within the hamster suprachiasmatic nucleus (SCN) and median eminence (ME) by quantitative autoradiography and compared the development of binding to the same regions in the rat. Binding in the hamster SCN was to a single class of sites (estimated Kd = 6 x 10(-10) M). Binding in the ME was approx. 1.5-fold that of the SCN. Full displacement of binding from the SCN and ME of adults and neonates was achieved by 10(-8) M IGF-I, 10(-8) M IGF-II or 10(-6) M insulin. Binding within the hamster SCN was evident by E15, peaked between P5 and P7 and decreased to adult levels by P20 while binding in the rat SCN peaked perinatally and declined to adult levels by P6. Binding in the hamster ME was evident at P4, peaked by P12 and decreased to adult levels by P20 while binding in rat ME was present by P2, peaked by P7 and declined to adult levels by P9. These results demonstrate a different developmental time course for [125I]IGF-I binding between the SCN and ME of hamster and rat. The peak binding in the SCN of each species correlates with previously reported time courses for onset of retinohypothalamic innervation of the SCN. Further study of IGFs in these regions may help elucidate the developmental role of brain IGFs.     

Characterization of [3H] CCK4 binding sites in mouse and rat brain

We have investigated the possible occurrence of distinct CCK8 and CCK4 binding sites in the brain by comparing the binding characteristics of [3H] CCK4 to those of the CCK8 analogue, [3H] Boc (Nle28,31]CCK27-33 (BDNL-CCK7). [3H] CCK4 and [3H] BNDL-CCK7 were shown to interact with mouse brain membranes with very similar maximal binding capacities 31.7 +/- 2.1 fmol/mg prot (KD = 3.78 +/- 0.47 nM) and 38.9 +/- 2.2 fmol/mg prot (KD = 0.26 +/- 0.02 nM) respectively. The apparent affinities of five CCK analogues for the sites labelled by both probes were almost identical. Autoradiographic studies revealed that the distribution of [3H] CCK4 binding sites in rat forebrain was the same as that of [3H] BDNL-CCK7, with high densities of receptors in the cortex, nucleus accumbens, olfactory bulb and the medial striatum, moderate densities in the amygdala, the hippocampus, several nuclei of the thalamus and hypothalamus. However in the interpenduncular nucleus where there was moderate binding of [3H]BDNL-CCK7, no [3H]CCK4 labelling was observed. These studies demonstrated the occurrence of one class of high affinity binding sites for [3H] CCK4 in mouse and rat brain, with characteristics similar to those already reported with CCK33, CCK8 and pentagastrin probes. Nevertheless the presence of a small amount of very high affinity binding sites for [3H]CCK4 cannot be excluded.     

Angiotensin II binding sites in the hamster brain: localization and subtype distribution

This study was designed to characterize the distribution of angiotensin II (AII) binding sites in the hamster brain. Brain sections were incubated with [¹²⁵I][sar¹, ile⁸]-angiotensin II in the absence and presence of angiotensin II receptor subtype selective compounds, losartan (AAT, subtype) and PD123177 (AT₂ subtype). Binding was quantified by densoritometric autoradiograms and localized by comparison with adjacent thionin stained sections. The distribution of AII binding sites was similar to that found in the rat, with some exceptions. [¹²⁵I][sar¹, ile⁸]-angiotensin II binding was not evident in the subthalamic nucleus and thalamic regions, inferior olive, suprachiasmatic nucleus, and piriform cortex of the hamster, regions of prominent binding in the rat brain. However, intense binding was observed in the interpeduncular nucleus and the medial habenula of the hamster, nuclei void of binding in the rat brain. Competition with receptor subtype selective compounds revealed a similar AII receptor subtype profile in brain regions where binding is evident in both species. One notable exception is the medial geniculate nucleus, predominately AT₁ binding sites in the hamster but AT₂ in the rat. Generally, the AII binding site distribution in the hamster brain parallels that of the other species studied, particularly in brain regions associated with cardiovascular and dipsogenic functions. Functional correlates for AII binding sites have not been elucidated in the majority of brain regions and species mismatches might provide clues in this regard.     

Effect of neonatal capsaicin treatment on cholecystokinin-(CCK8) satiety and axonal transport of CCK binding sites in the rat vagus nerve

Cholecystokinin (CCK) binding sites which accumulate at ligatures placed on the rat vagus nerve may mediate the satiety actions of CCK. Treatment of neonatal rats with capsaicin attenuated the satiety effect of injected CCK in adult life. Capsaicin pretreatment also reduced, but did not eliminate, the accumulation of CCK binding sites proximal and distal to ligatures on either cervical trunk. A similar effect was observed following ligation of subdiaphragmatic vagal trunks. The CCK receptor antagonists, MK-329 and L-365,260, inhibited binding to capsaicin- and vehicle-treated nerves to a similar degree. Densities of CCK binding sites in the nucleus tractus solitarius and area postrema were also markedly affected by neonatal capsaicin treatment.     

Cholecystokinin and neuropeptide Y receptors on single rabbit vagal afferent ganglion neurons: site of prejunctional modulation of visceral sensory neurons

A [¹²⁵I]cholecystokinin (CCK) analog and [¹²⁵I]peptide YY (PYY) were used to localize and characterize CCK and neuropeptide Y (NPY) receptor binding sites in the rabbit vagal afferent (nodose) ganglion. High concentrations of CCK and NPY binding sites were observed in 10.6% and 9.2% of the nodose ganglion neurons, respectively. Pharmacological experiments using CCK or NPY analogs suggest that both subtypes of CCK (CCK-A and CCK-B) and NPY (Y1 and Y2) receptor binding sites are expressed by discrete populations of neurons in the nodose ganglion. These results suggest sites at which CCK or NPY, released in either the nucleus of the solitary tract or a peripheral tissue, may modulate the release of neurotransmitters from a select population of visceral primary afferent neurons. Possible functions mediated by these receptors include modulation of satiety, opiate analgesia, and the development of morphine tolerance.     

Topographical representation of the peripheral nerve branches of the facial nucleus of the opossum: a study utilizing horseradish peroxidase

The main facial nucleus of a marsupial, the North American opossum (Didelphis marsupialis virginiana), was subdivided into 6 portions by localizing HRP-positive neurons after injecting all muscles supplied by each major peripheral motor branch of the facial nerve. In the medial lobe of this dumbbell-shaped nucleus the caudal auricular nerve, rostral auricular ramus and cervical ramus were represented dorsemedially, dorsolaterally and ventrally, respectively. The lateral lobe contained zygomatic ramus cells dorsemedially, marginal mandibular ramus cells ventromedially and buccal rami cells laterally. The cells supplying the caudal digastric muscle were in the accessory facial nucleus. Thus, even though the facial nucleus of the opossum lacks distinct ramal subdivisions in Nissl preparations, such are evident after HRP labeling.     

The regional and subcellular development of cholecystokinin immunoreactivity in vertebrate brain

We have previously demonstrated that the development of CCK in rat brain occurs during the first postnatal month. In order to determine whether the appearance of CCK is associated with specific aspects of brain histogenesis, we examined the development of brain CCK immunoreactivity in both precocial and altricial mammals and birds. The two precocial species (guinea pig and chicken) were found to achieve adult CCK concentrations prenatally, while the altricial species (zebra finch and rat) manifested adult brain CCK concentrations only after several weeks of postnatal development. In adulthood, both mammals showed relatively high forebrain CCK concentrations, while the two species of birds manifested much lower forebrain levels. Brainstem levels of CCK were similar in all species studied. In each species, the development of CCK followed a common time course across all major brain areas, although adult brainstem levels of CCK were generally attained shortly before adult forebrain levels. Correlation of our comparative ontogenetic data with known patterns of brain histogenesis indicated that CCK development follows regional neuroblastic proliferation, migration and differentiation, and occurs during or soon after local synaptogenesis. In the rapidly developing precocial chicken brain, CCK production precedes the postnatal gliogenic and myelinogenic increases in brain weight, suggesting that neurogenic production of CCK occurs independently of these non-neuronal maturation events. Subcellular fractionation of developing chicken brain revealed that a substantial fraction of brain CCK is localized in synaptosomes relatively early in embryogenesis; this synaptosomal localization becomes even more pronounced with further brain maturation. This early appearance of CCK in synaptic terminals indicates a correspondingly precocial maturation for the intraneuronal mechanisms subserving peptide cleavage, axonal transport and vesicular insertion, and suggests that CCK may be available for neurotransmission quite early in development. In an analysis of the molecular forms of CCK, gel filtration disclosed no differences between species or different brain areas in the form of CCK present. CCK-8 always predominated in brain, with smaller void volume (pro-CCK) peaks, and negligible amounts of CCK-33. Finally, duodenal CCK (largely CCK-33) appeared much earlier than brain CCK in all species examined, suggesting that the gut and brain CCK systems develop independently of one another.