Theresa A. Harrison

Hypothalamic orexin A-immunoreactive neurons project to the rat dorsal medulla

Retrograde tract tracing combined with immunohistochemical techniques were used to identify the origin of orexin A-immunoreactive (OrA-ir) fibers in the rat medulla. One to 5 days following injection of the fluorescent dye Fluorogold into the dorsal medulla, labeled neurons were found in the lateral half of the lateral hypothalamus, paraventricular, perifornical, dorsomedial, dorsal and posterior hypothalamic nuclei. Labeling the same sections with OrA antisera revealed a concentration of OrA-ir neurons in the perifornical and dorsomedial regions of the tuberal hypothalamus. A maximum of 10% of Fluorogold-labeled hypothalamic neurons were OrA-ir and 15% of OrA-ir hypothalamic neurons contained Fluorogold. Our results demonstrate that a fraction of OrA-ir neurons in the tuberal hypothalamus project to areas of the medulla that are involved in autonomic functions.

Short-term plasticity in primary somatosensory cortex of the rat: rapid changes in magnitudes and latencies of neuronal responses following digit denervation

Recordings were made from neurons in primary somatosensory (SmI) forepaw cortex of rats to study the time course of changes in responses beginning immediately following denervation (ligation) of a single digit. Before denervation, neuronal receptive fields (RFs) defined by tactile stimulation varied in size from small regions of one digit to larger areas covering several digits and palmar pads. With electrical stimulation, most neurons responded best to one (on-focus) digit and less to other (off-focus) digits; on-focus stimulation yielded more spikes per stimulus and shorter spike latencies (Lmin) than did off-focus stimulation. After ligation of the on-focus digit, most neurons showed increased responsiveness to stimulating one or several off-focus digits and palmar regions of the forepaw: (1) tactile stimulation showed that the RFs of all but one neuron expanded to include previously “ineffective” skin regions, such as digits or palmar pads adjoining the original RF; (2) electrical stimulation usually evoked stronger responses from neighboring off-focus digits and sometimes elicited novel responses from previously ineffective digits — seven of ten neurons showed increases in spikes per stimulus, which tended to approach stable values within 60–90 min after denervation; three of ten neurons showed decreases in Lmin with time, but most revealed no significant changes. These results suggest that dynamic response properties, as well as RFs, of SmI cortical neurons can be modified rapidly by blocking afferent input from dominant on-focus skin regions. RFs expand and novel responses appear, with concomitant increases in response magnitude and, in some cases, decreases in response latency over time. These findings seem to reflect a rapid increase in synaptic efficacy of weak or previously ineffective inputs from cutaneous afferent nerve fibers.

Distribution of Cocaine- and Amphetamine-Regulated Transcript Peptide in the Guinea Pig Intrinsic Cardiac Nervous System and Colocalization With Neuropeptides or Transmitter Synthetic Enzymes

This study was conducted to establish the presence of cocaine‐ and amphetamine‐regulated transcript peptide (CARTp) immunoreactivity in neurons and fibers within guinea pig atrial whole‐mount preparations containing the intrinsic cardiac ganglia. Many cardiac ganglia, but not all, in a given whole‐mount preparation, were innervated by CARTp‐immunoreactive (IR) fibers. Following explant culture of whole mounts for 72 hours, the CARTp‐IR fiber networks were absent, but the number of CARTp‐IR neurons was increased markedly. These observations suggested that the majority of the CARTp‐IR fibers in the intracardiac ganglia were derived from sources extrinsic to the heart. In control whole‐mount preparations, very few CARTp‐positive neurons were present. The few intrinsic CARTp‐IR neurons also exhibited choline acetyltransferase (ChAT) immunoreactivity, indicating that they make up a small subpopulation of cholinergic postganglionic neurons. Some CARTp‐IR neurons also exhibited nitric oxide synthase (NOS) immunoreactivity, indicating that they were nitrergic as well. We compared the immunohistochemical staining patterns of CARTp‐IR fibers with the staining patterns of a number of other neurotransmitters or neurotransmitter synthetic enzymes that mark specific extrinsic inputs. The CARTp‐IR fibers were not immunoreactive for ChAT, tyrosine hydroxylase, calcitonin gene‐related peptide, or substance P. However, virtually all CARTp‐IR fibers exhibited immunoreactivity to neuronal NOS (a marker for nitric oxide‐producing neurons). CARTp‐IR cells and NOS‐IR cells were present in the nodose ganglia. In addition, CARTp‐IR neurons in the nodose also were stained positively for NADPH‐diaphorase. Thus, we propose that most CARTp‐IR fibers within the guinea pig intrinsic cardiac ganglia are vagal afferent fibers that also contain NOS. J. Comp. Neurol. 439:73–86, 2001.

Compensatory responses and development of the nodose ganglion following ablation of placodal precursors in the embryonic chick (Gallus domesticus).

The nodose ganglion is the distal cranial ganglion of the vagus nerve which provides sensory innervation to the heart and other viscera. In this study, removal of the neuronal precursors which normally populate the right nodose ganglion was accomplished by ablating the right nodese placode in stage 9 chick embryos. Subsequent histological evaluation showed that in 54% of lesioned embryos surviving to day 6, the right ganglion was absent. Most embryos surviving to day 12, however, had identifiable right ganglia. In day 12 embryos, the right ganglion which developed was abnormal, with ganglion volume and ganglion cell diameter reduced by 50% and 20%, respectively, compared to control ganglia. To investigate the source of the neuron population in the regenerated ganglion, we combined nodose placode ablation with bilateral replacement of chick with quail “cardiac” neural crest (from mid-otic placode to somite 3). These cells normally provide only non-neuronal cells to the nodose ganglion, but produce neurons in other regions. At day 9, quail-derived neurons were identified in the right nodose ganglia of these chimeras, indicating that cardiac neural crest cells can generate neurons in the ganglion when placode-derived neurons are absent or reduced in number. On the other hand, we found that “sympathetic” neural crest (from somites 10 to 20) does not support ganglion development, suggesting that only neural crest cells normally present in the ganglion participate in reconstituting its neuronal population. Our previous work has shown that right nodose placode ablation produces abnormal cardiac function, which mimics a life-threatening human heart condition known as long QT syndrome. The present results suggest that the presence of neural crest-derived neurons in the developing right nodose ganglion may contribute to the functional abnormality in long QT syndrome.

Chorda tympani nerve stimulation evokes Fos expression in regionally limited neuron populations within the gustatory nucleus of the solitary tract.

The distribution of neurons in the rostral nucleus of the solitary tract (rNST) that respond to gustatory input from the anterior tongue was visualized by Fos protein immunohistochemistry following electrical stimulation of the chorda tympani (CT) nerve in rats. Maps of Fos-immunoreactive (Fos-ir) neurons were compared with the distribution of CT afferent terminal fields labeled by transganglionic transport of rhodamine-dextran in a separate group of animals. The primary concentration of Fos-ir neurons localized in register with the major terminal fields of CT afferent fibers, in the central third of the rostral 1.0 mm of the NST ipsilateral to the stimulated nerve. A similar correspondence in location and degree of labeling of Fos-ir neurons and afferent terminals was observed in the ipsilateral dorsal spinal trigeminal complex (Sp5) pars caudalis, near the obex, and the Sp5 pars oralis near the rostral pole of the rNST. Thus, the magnitude of Fos upregulation in brainstem targets of the CT nerve having chemosensory or nociceptive function, was proportional to the relative density of the CT afferent input. This correspondence, and the absence of labeling in neurons known to be one additional synapse away from the afferent input within gustatory or oral reflex pathways, suggests that the cell map obtained represents mainly neurons that are directly activated via primary afferent synapses from CT fibers. The availability of a method to histochemically identify a population of putative second-order taste neurons will facilitate analysis of the cellular/molecular properties of these neurons and of synaptic circuitry in the rNST.

Distinct regional distributions of NK1 and NK3 neurokinin receptor immunoreactivity in rat brainstem gustatory centers

Tachykinins and their receptors are present in gustatory centers, but little is known about tachykinin function in gustation. In this study, immunohistochemical localization of substance P and two centrally prevalent neurokinin receptors, NK1 and NK3, was carried out in the rostral nucleus of the solitary tract and the caudal parabrachial nucleus to evaluate regional receptor/ligand correspondences. All three proteins showed regional variations in labeling density that correlated with distinct sites in gustatory centers. In the rostral nucleus of the solitary tract, the relative densities of substance P and NK1 receptors varied in parallel across subnuclei, with both being moderate to dense in the dorsocentral, chemoresponsive zone. NK3 receptors had a distinct distribution in the caudal half of this zone, suggesting a unique role in processing taste input from the posterior tongue. In the caudal parabrachial nucleus, substance P and NK1 receptor immunoreactivities were dense in the pontine taste area, while NK3 receptor labeling was sparse. The external medial subnucleus had substantial NK3 receptor and substance P labeling, but little NK1 receptor immunoreactivity. These findings suggest that distinct tachykinin ligand/neurokinin receptor combinations may be important in local processing of information within brainstem gustatory centers.

Role of the levator palpebrae superioris (LPS) muscle in effecting nictitating membrane movement in the rabbit.

Abstract Examination of the origin and course of the muscle fibers attached to the nictitating membrane (NM) of the rabbit demonstrated that these muscle fibers constitute part of the nasal branch of the levator palpebrae superioris (LPS) muscle. Other fibers of the nasal branch of the LPS muscle were observed to terminate in the nasal half of the upper eyelid. No further anatomical division of the nasal LPS into distinct eyelid and NM branches was observable by dissection. Contraction of the fibers inserting in the NM, produced by direct electrical stimulation, resulted in movement of the extended membrane towards its resting position in the corner of the eye. The observed anatomical relationships demonstrate in part that functional control of NM movement is structurally linked to control of upper eyelid elevation in this species. The origin and distribution of fibers inserting in the NM, and the effects of contraction of the muscle fibers on movement of the NM, indicate that contraction of these fibers could both oppose extension of the NM and/or initiate or contribute to retraction of the membrane following extension. Because of their position and the direction of NM movement caused by their contraction, these muscle fibers cannot actively cause or contribute to extension of the NM. Therefore, it is suggested that the LPS has a role in effecting movement of the rabbit NM by actively contributing to its retraction or reopening.

Developmental Characteristics of the Chick Nodose Ganglion

Morphometric studies were carried out on the chick nodose ganglion between day 5 of incubation and 2 weeks after hatching. Previous findings showed that ablation of the nodose placode, the locus of precursor cells of nodose ganglion sensory neurons, results in abnormal cardiac function, and that these precursors can be induced to migrate abnormally to the heart and express abnormal phenotypes there, following cardiac neural crest ablation. These results prompted us to investigate further the normal development of nodose ganglion neurons. We find that the major period of neuron generation from placodal precursor cells in the ganglion occurs prior to day 5 of incubation. The loss of more than half of these neurons takes place between embryonic days 5 and 20, while neuron and ganglion sizes increase dramatically. Myelination is not seen at day 12 of incubation, but is present on day 15. Neurons continue to develop after hatching (day 21), reaching their adult size by 2 weeks after hatching. Unexpectedly, we found that the number of neurons increases after hatching, reaching the adult level of 62% more than embryonic day-20 numbers by 2 weeks after hatching. The mechanisms underlying the increase in neuron number after hatching are unexplained and require further investigation.

Accelerated turnover of taste bud cells in mice deficient for the cyclin-dependent kinase inhibitor p27Kip1

BackgroundMammalian taste buds contain several specialized cell types that coordinately respond to tastants and communicate with sensory nerves. While it has long been appreciated that these cells undergo continual turnover, little is known concerning how adequate numbers of cells are generated and maintained. The cyclin-dependent kinase inhibitor p27Kip1 has been shown to influence cell number in several developing tissues, by coordinating cell cycle exit during cell differentiation. Here, we investigated its involvement in the control of taste cell replacement by examining adult mice with targeted ablation of the p27Kip1 gene.ResultsHistological and morphometric analyses of fungiform and circumvallate taste buds reveal no structural differences between wild-type and p27Kip1 -null mice. However, when examined in functional assays, mutants show substantial proliferative changes. In BrdU incorporation experiments, more S-phase-labeled precursors appear within circumvallate taste buds at 1 day post-injection, the earliest time point examined. After 1 week, twice as many labeled intragemmal cells are present, but numbers return to wild-type levels by 2 weeks. Mutant taste buds also contain more TUNEL-labeled cells and 50% more apoptotic bodies than wild-type controls. In normal mice, p27 Kip1 is evident in a subset of receptor and presynaptic taste cells beginning about 3 days post-injection, correlating with the onset of taste cell maturation. Loss of gene function, however, does not alter the proportions of distinct immunohistochemically-identified cell types.Conclusionsp27Kip1 participates in taste cell replacement by regulating the number of precursor cells available for entry into taste buds. This is consistent with a role for the protein in timing cell cycle withdrawal in progenitor cells. The equivalence of mutant and wild-type taste buds with regard to cell number, cell types and general structure contrasts with the hyperplasia and tissue disruption seen in certain developing p27Kip1 -null sensory organs, and may reflect a compensatory capability inherent in the regenerative taste system.

Consequences of low or moderate prenatal ethanol exposures during gastrulation or neurulation for open field activity and emotionality in mice.

In a previous study we used a mouse model for ethanol exposure during gastrulation or neurulation to investigate the effects of modest and occasional human drinking during the 3rd or 4th week of pregnancy (Schambra et al., 2015). Pregnant C57Bl/6J mice were treated by gavage during gastrulation on gestational day (GD) 7 or neurulation on GD8 with 2 doses 4h apart of either 2.4 or 2.9g ethanol/kg body weight, resulting in peak blood ethanol concentrations (BECs) of 104 and 177mg/dl, respectively. We found that mice exposed to the low dose on either day were significantly delayed in their neonatal sensorimotor development. In the present study, we tested the same cohort of mice in an open field as juveniles on postnatal day (PD) 23-25 and as young adults on PD65-67 for prenatal ethanol effects on exploration and emotionality with measures of activity, rearing, grooming and defecation. We evaluated the effects of dose, sex, day of treatment and day of birth by multiple regression analyses. We found that, compared to the respective gavage controls, juvenile mice that had been prenatally exposed to the low BEC on either GD7 or GD8 were significantly hypoactive on the first 2 test days, reared significantly more on the last 2 test days, and groomed and defecated significantly more on all 3 test days. Only mice that had been treated on GD7 remained hypoactive as adults. Juvenile mice prenatally exposed to the moderate BEC on GD7 groomed significantly more, while those exposed on GD8 reared and defecated significantly more. Sex differences were highly significant in adult control mice, with control males less active and more emotional than females. Similar, but smaller, sex differences were also evident in adults exposed to ethanol prenatally. Persistence into later life of a deleterious effect of premature birth (i.e., birth on GD19 rather than GD20) on weight and behavior was not consistently supported by these data. Importantly, mice shown previously to be delayed in sensorimotor development as neonates, in the present study demonstrated hypoactivity and increased emotionality in open field behaviors as juveniles, and those mice exposed during gastrulation remained hypoactive as adults. Thus, we propose that the delayed motor development, hypoactivity and emotionality we observed in mice exposed to a low BEC during gastrulation or neurulation may relate to an attention deficit-activity disorder in humans, possibly the inattentive subtype, or Sluggish Cognitive Tempo (SCT). We further discuss concerns about occasional light or moderate alcohol consumption during the 3rd or 4th week of human pregnancy.