Hypothetical anatomical model to describe the aberrant gag reflex observed in a clinical population of orally deprived children.

In this 'clinical conundrum', we propose a hypothetical anatomical model to explain the abnormal gag reflex that is consistently observed in a clinical population of children experiencing feeding delays. This model is based on the presence of 'transient' connections formed during the normal development of autonomic brainstem circuitry involving the nucleus tractus solitarius (NTS). We propose that, as a result of normal feeding and swallowing, the activity of these transient fibers typically diminishes shortly after birth. In children who are orally deprived during infancy, these transient connections persist and the aberrant gag reflex is maintained into childhood. The most critical feature of the proposed model is the idea that swallowing during feeding initiates the retraction of the tactile 'transient' input to NTS. In the NICU feeding clinics, it has been suggested that triggering the gag reflex in neonates by tactile stimulation of non-oral body areas and anterior portions of the mouth directly or indirectly may contribute to oral feeding delays. To the contrary, we propose an anatomical model to suggest that oral feeding delays and lack of swallowing food, when experienced by neonates, actually contribute to the development of the aberrant gag reflex observed in later developmental stages.

The effects of deafferentation and exogenous NGF on neurotrophins and neurotrophin receptor mRNA expression in the adult superior cervical ganglion.

Levels of nerve growth factor (NGF) and neurotrophin-3 (NT-3) protein and neurotrophin receptor mRNA in adult sympathetic neurons were investigated following surgical removal of preganglionic input and/or in vivo administration of NGF. Expression of trkC and p75, but not trkA, was significantly decreased following a 3-week deafferentation of the superior cervical ganglion (SCG). Protein levels of NGF and NT-3 in the SCG were unchanged by deafferentation. A 2-week intracerebroventricular infusion of NGF without deafferentation resulted in enhanced mRNA levels of trkA, trkC, and p75 as well as significantly increased NGF and NT-3 protein in the SCG. When NGF infusion followed deafferentation, both trkA and p75 showed significant increases while trkC levels were similar to control values. NGF protein was not increased in the SCG when deafferentation preceded exogenous NGF, yet NT-3 was elevated and levels were similar to cases receiving NGF infusion only. These results support a role for preganglionic input in trkC and p75 expression in adult sympathetic neurons. The increased levels of NT-3 protein and trkC gene expression observed following NGF infusion suggest that NGF influences NT-3 regulation in adult sympathetic neurons. In addition, the present findings provide evidence that, when preganglionic input is removed prior to the NGF infusion, NT-3 effectively competes with NGF for trkA binding. Taken together, we propose that NT-3 may play a role in the robust sprouting of sympathetic cerebrovascular axons previously observed following NGF administration, particularly when deafferentation precedes the NGF infusion period.

Sympathetic ingrowth to the trigeminal ganglion following intracerebroventricular infusion of nerve growth factor.

The objective of the present study was to examine the remodeling of uninjured sympathetic axons in the adult rat trigeminal ganglion following a 2-week in vivo intracerebroventricular infusion of NGF. The accumulation of infused NGF in the trigeminal was assessed using ELISA and sympathetic fibers were localized immunohistochemically with an antibody to tyrosine hydroxylase (TH). In addition, high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) allowed for biochemical measurements of the catecholamines norepinephrine (NE) and dopamine (DA). Increased NGF protein in the trigeminal ganglion was paralleled by a significant increase in sympathetic fibers and pericellular plexuses (i.e. baskets) in the cell body regions. Some ganglia showed elevated NE following NGF infusion, yet the 88% increase in mean NE did not reach significance. Following bilateral removal of the sympathetic superior cervical ganglia (SCG), a significant reduction was observed in overall NE levels and in TH-immunoreactive (-ir) fibers in the cell body regions and peripheral branches, suggesting the SCG as the origin of the sympathetic ingrowth. However, mean DA levels as well as TH-ir fibers within the trigeminal central branch were unaffected by NGF infusion or removal of the SCG and likely resulted from intrinsic dopaminergic cell bodies. In conclusion, our data provide evidence that the increased availability of NGF in the young adult rat trigeminal ganglion observed following in vivo NGF infusion enhanced sympathetic associations with the sensory neurons in the trigeminal, supporting a role for NGF in the regulation of sympathosensory interactions.

Evidence that nerve growth factor mediates the formation of sensory pericellular baskets in the rat trigeminal ganglion.

A role for nerve growth factor (NGF) in the remodeling of sensory neurons in the trigeminal ganglion was examined. Intracerebroventricular NGF infusion and/or bilateral removal of the sympathetic superior cervical ganglia, both of which are believed to increase the availability of NGF to primary sensory neurons, resulted in a significant increase in the frequency of calcitonin gene-related peptide immunoreactive pericellular baskets. The results of this study suggest that increased NGF is sufficient to enhance the formation of sensory baskets in this ganglion, and provide evidence that NGF may mediate the formation of sensory baskets in the sensory ganglia following injury.

Evidence for reduced accumulation of exogenous neurotrophin by aged sympathetic neurons.

The present study investigated the potential for neurotrophin uptake by cerebrovascular axons and subsequent accumulation in the aged superior cervical ganglion (SCG) following a two week intracerebroventricular infusion of nerve growth factor (NGF). In the SCG from aged rats, NGF protein levels declined significantly compared with the SCG from young adult rats. Following NGF infusion, perivascular axons from both young adult and aged rats showed intense NGF immunostaining. In addition, significant increases in NGF protein were shown using enzyme-linked immunosorbent assay (ELISA) and in counts of NGF immunopositive cell bodies in the SCG when compared with age-matched controls. NGF accumulation in ganglia from aged rats, however, was significantly less when compared with ganglia from young adult rats. The results of the present study suggest that NGF protein is significantly reduced in aged ganglia with the neurons retaining some capacity to take up and transport exogenous neurotrophin. Even so, the potential for NGF accumulation is dramatically reduced in aged rats when compared with that of young adult rats. While previous results have shown robust NGF-induced neurotransmitter responses by sympathetic neurons from the aged animal, the present finding of reduced accumulation of NGF in aged sympathetic neurons suggests an age-related difference in the utilization or transport of NGF.

Plasticity of aged perivascular axons following exogenous NGF: analysis of catecholamines.

The present study investigated the atrophy of aged perivascular sympathetic axons and the response of these cerebrovascular neurons to the neurotrophin nerve growth factor (NGF). Using high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) to quantify catecholamines and immunohistochemical methods to quantify the density of TH immunoreactive fibers, we found a significant decrease in norepinephrine (NE) and TH in aged sympathetic axons. However, following in vivo administration of exogenous neurotrophin, aged neurons exhibited a robust response to NGF that was similar to the young adult, suggesting little decline in the capability of aged neurons to utilize exogenous neurotrophin. These results suggest that the age-related atrophy of aged sympathetic axons may result primarily from reduced availability of target-derived neurotrophin rather than from intrinsic alterations of neuronal function.

Statistical comparison of axon-scaled neurochemical production.

Treatments designed to increase neurochemical levels may also result in increases in the numbers of axons that produce the neurochemicals of interest. A natural research question is how does one compare the average neurochemical production per axon between two (or more) experimental groups. Two statistical methods are proposed for this problem. The first method utilizes a delta-method approximation to the variance of a function of random variables while the second method is based on the bootstrap. These methods are illustrated with data obtained from perivascular norepinephrine following intracerebroventricular infusion of neurotrophin nerve growth factor in adult rats and are studied in a small simulation experiment. The delta-method confidence intervals exhibited better coverage properties than the bootstrap alternative.

Remodeling of adult sensory axons in the superior cervical ganglion in response to exogenous nerve growth factor.

In previous studies, we found that a 2-week in vivo intracerebroventricular infusion of nerve growth factor (NGF) elicited a sprouting response by sympathetic perivascular axons associated with the intradural segment of the internal carotid artery. We hypothesized that NGF infused into the ventricular system would be internalized by responsive sympathetic cerebrovascular axons, retrogradely transported to parent cell bodies in the superior cervical ganglion (SCG), and subsequently released into the local ganglionic environment. Because fibers exhibiting immunoreactivity for calcitonin gene related peptide (CGRP) have been localized in the SCG, we used immunohistochemical methods to investigate whether a response by CGRP-immunoreactive axons in the SCG occurred following the proposed transport to and release of exogenous NGF in the ganglion. In consecutive tissue sections of the SCG stained for either CGRP or NGF, we found CGRP pericellular 'baskets' surrounding identified NGF-immunoreactive cell bodies. Nerve growth factor infusion resulted in a significant increase both in the number of CGRP pericellular baskets and in NGF-immunoreactive cell bodies. A significant positive correlation (r=0.95, P<0.05) between the pericellular baskets and NGF-immunoreactive cell bodies was observed, suggesting that intracranial projection neurons in the SCG released infused NGF (or possibly a converted signal) into the local ganglionic environment to elicit remodeling of CGRP fibers to form pericellular baskets. These findings were confirmed in sections double labeled for NGF and CGRP immunoreactivity. This remodeling suggests that exogenous NGF may mediate retrograde transneuronal plasticity, allowing for future in vivo examinations of the mechanisms involved in neurotrophin transport and release.

Uninjured aged sympathetic neurons sprout in response to exogenous NGF in vivo.

The extent to which the loss of plasticity by aged neurons is due to changes in the neuronal environment or to a loss of growth potential of the neurons has not been determined. In previous studies we observed that young adult cerebrovascular axons undergo a sprouting response following a 2-week intracerebroventricular infusion of nerve growth factor (15 microg; NGF). The present study used electron microscopy to examine the innervation of the intradural segment of the internal carotid artery of the aged rat and to determine whether aged sympathetic perivascular axons would respond to in vivo infusion of NGF. Young adult and aged Fischer 344 female rats received a 2-week intracranial infusion of NGF (15 microg) or vehicle (VEH) and were perfused for electron microscopy. Although there was no change in the total number of perivascular axons associated in aged VEH when compared with young adult VEH, a significant reduction was observed in aged VEH when total axons and sympathetic axons were expressed per microm2 vascular wall, reflecting an age-related increase in blood vessel size. Following NGF infusion, aged sympathetic axons were significantly increased by 192% compared with aged VEH cases. These results suggest that there is a proportional reduction in sympathetic cerebrovascular neurons with aging but that they exhibit robust sprouting in response to an exogenous neurotrophin.

Dose-dependent response of mature cerebrovascular axons in vivo following intracranial infusion of nerve growth factor.

The perivascular axons associated with the intradural segment of the internal carotid artery undergo a sprouting response following intracerebroventricular infusion of nerve growth factor (NGF). The objective of the present study was to determine whether a relationship exists between the number of sprouted axons and the amount of NGF infused into the ventricular system. Using regression analysis, we observed a significant log-log relationship between the dose of NGF and number of axons. No significant relationship was observed for the control (VEH) group. A significant increase in axonal number was observed following infusion of 3.0 micrograms NGF and higher when compared with VEH treatment of similar concentration. Results of this analysis suggest that a maximal response to NGF is approximated at doses of 15 micrograms or higher. These findings suggest a dose-dependent relationship between the response of mature sympathetic cerebrovascular axons in vivo and the dose of exogenous NGF.

Sympathetic response to intracranial NGF infusion in the absence of afferent input: axonal sprouting without neurotransmitter production.

The anatomical relationships between postganglionic sympathetic neurons, their targets, and their afferent inputs provide an opportunity to experimentally distinguish between the anterograde and the retrograde influences on neuronal responsivity to growth factors. In the present study, the effect of decentralization of the superior cervical ganglion (SCG) on the NGF-induced sympathetic sprouting response by mature cerebrovascular axons was assessed in young adult rats. Growth by the perivascular axons associated with the intradural segment of the internal carotid artery was quantified using electron microscopy and changes in norepinephrine (NE) levels were monitored using high-performance liquid chromatography coupled with electrochemical detection. The mean number of perivascular axons observed in the treatment group receiving decentralization of the SCG prior to NGF infusion was not significantly different from that in NGF-infused cases, suggesting that central input was not required for axonal growth of intact sympathetic neurons. However, decentralization prevented the typical NGF-induced increase in perivascular NE associated with the ICA, indicating that afferent input was necessary for the neurotransmitter increase to occur. Thus, afferent input appears to play a role in the regulation of neurotransmitter expression of the sprouted axons but is not required for trophic factor-induced axonal growth.

Increased perivascular norepinephrine following intracerebroventricular infusion of NGF into adult rats.

In the present study, we used high performance liquid chromatography coupled with electrochemical detection to examine perivascular catecholamines associated with the intradural segment of the internal carotid artery following a 2-week in vivo intracerebroventricular infusion of the neurotrophin nerve growth factor (NGF). Following administration of NGF, a significant increase (87.3%) in perivascular norepinephrine (NE; microgram/g) was observed when compared with vehicle-infused controls, suggesting that increased sympathetic neurotransmitter accompanies the NGF-induced sprouting response by sympathetic perivascular axons previously observed using electron microscopy (13, 15). The biochemical quantification of perivascular NE in the present study taken together with our previous morphological quantification of perivascular sprouts at the ultrastructural level reveal that the increase in NE is not proportional to the increase in the number of axons. Thus, when compared with controls, the relative amount of norepinephrine per axon apparently is reduced following NGF infusion. The apparent decrease in NE per axon following NGF infusion suggests that, during the 2-week infusion period, exogenous NGF did not stimulate the biosynthesis of perivascular NE beyond that necessary to accommodate the newly sprouted axons. These results extend our morphological findings and provide evidence for plasticity of neurotransmitter expression by adult sympathetic perivascular axons in vivo. In addition, we provide evidence that the increased perivascular catecholamine histofluorescence previously observed following NGF infusion results from an increase in the number of perivascular axons associated with the vessel rather than from an increase in the amount of NE within individual axons.

Plasticity of mature sensory cerebrovascular axons following intracranial infusion of nerve growth factor.

Mature perivascular sympathetic axons associated with the intradural segment of the internal carotid artery (ICA) of the adult rat respond by sprouting following a two week infusion of nerve growth factor (NGF) into the lateral ventricle of the brain. Because nonsympathetic axons such as those comprising the sensory and parasympathetic population have been shown to respond to NGF, the present study was carried out to determine whether mature sensory axons respond to in vivo NGF infusion and whether competitive interactions between the innervating populations might affect the responsiveness of these axons to NGF. Standard electron microscopic techniques as well as calcitonin-gene-related peptide (CGRP) immunohistochemistry at the light microscopic level were used to examine the effects of intracerebroventricular NGF infusion on mature perivascular fibers with and without prior sympathetic denervation (i.e., bilateral superior cervical ganglionectomy). Following NGF infusion, CGRP-immunoreactive fibers appeared thicker and more numerous in the longitudinal plane when compared with vehicle controls. However, at the ultrastructural level, a significant increase in the total number of axons was not observed, although there was an increase in the number of large granular vesicles, suggesting that the CGRP fibers responded to exogenous NGF with an increase in neurotransmitter content, but not by sprouting. Sympathetic denervation, on the other hand, resulted in a significant increase in the number of fibers passing in the circumferential plane. The most dramatic change in CGRP immunoreactivity was observed following combined sympathetic denervation and subsequent NGF infusion, where, in addition to the presence of thicker immunoreactive fibers, a significant increase in the perivascular density of immunoreactive fibers associated with the intradural blood vessels was observed. These findings suggest that exogenous NGF has different effects on mature sympathetic and nonsympathetic fibers that innervate intradural blood vessels. The former exhibit robust sprouting, whereas the latter do not sprout in response to NGF but show evidence for increased neuropeptide content. In addition, the heightened response by sensory axons following denervation and subsequent NGF infusion provides support for the idea that sensory and sympathetic axons normally compete for target space and/or target-derived neurotrophic factors.

The duration of sprouted cerebrovascular axons following intracranial infusion of nerve growth factor.

We reported previously that a 2-week infusion of the trophic protein nerve growth factor (NGF) into the lateral ventricle of the adult rat brain elicits a sprouting response by perivascular axons associated with the intradural segment of the internal carotid artery. In the present study, we used electron microscopy to determine whether the sprouted axons persist following cessation of NGF delivery and, if not, to determine the time course of their disappearance. Our results demonstrate that NGF-induced sprouted axons do not persist following cessation of NGF delivery. The total number of axons at 1 week following the end of the NGF infusion was elevated compared to control values, but significantly reduced compared with NGF cases sacrificed immediately following the infusion period. Three weeks following the end of the NGF infusion, the total number of axons was similar to controls although there were no signs of axonal degeneration. These results suggest that continued elevation of NGF levels is necessary to maintain the sprouted axons and that endogenous levels of NGF, or other factors produced by the vascular target tissue, are not sufficient to maintain the newly formed axons. The demonstration that mature perivascular axons proliferate and disappear as a function of exogenous NGF exposure supports the accumulating evidence for continued plasticity in the mature nervous system.

Thalamic afferents of area 4 and 6 in the dog: a multiple retrograde fluorescent dye study.

In the present study, we compared the distribution of thalamocortical afferents of cortical area 4 to that of cortical area 6 in the dog, using fluorescent tracers. Multiple injections of combinations of two dyes (diamidino yellow dihydrochloride, Evans blue, fast blue, granular blue) were made into either the anterior and posterior sigmoid gyri or into the medial and lateral regions of the anterior sigmoid gyrus in the anesthetized dog. We found that the thalamic afferents of areas 4 and 6 arise from topographically organized bands of cells that traverse several thalamic nuclei and extend throughout the rostrocaudal extent of the thalamus. The most medial band included area 6-projecting neurons in the anterior nuclei, the rhomboid nucleus, the ventral anterior nucleus (VA), ventromedial nucleus (VM) and mediodorsal nucleus (MD). Within this band, cells projecting to medial area 6 a alpha tended to be more numerous in the anterior nuclei, anterior parts of VA and VM and anterior and caudal parts of MD. Fewer cells in MD but more cells in caudal parts of VA and VM projected to lateral area 6 a beta. Lateral bands of cells in central through lateral parts of VA and VL projected topographically to lateral area 4 on the anterior sigmoid gyrus and lateral through medial parts of postcruciate area 4. The most lateral band of cells in VL continued ventrally into the zona incerta. Area 4 also received input from VM and the central lateral (CL) and centrum medianum (CM) nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve growth factor-induced sprouting of mature, uninjured sympathetic axons.

The infusion of nerve growth factor (NGF) into the lateral ventricle of the mature rat brain elicits a sprouting response from axons associated with the intradural segment of the internal carotid artery. Using electron microscopic techniques, we observed a three-fold increase in the total number of perivascular axons. This NGF-elicited response is characterized by a dramatic reduction in glial cell ensheathment similar to that observed during development and by the presence of profiles devoid of organelles that may represent newly formed sprouts. In spite of the increase in axon number, no significant changes in the percentage of small, medium, or large axons were observed. The three-fold increase in the total number of axons was accompanied by an increase in the number of axons/fascicle but no change in the number of fascicles. This, along with the observation that a majority of sprouted axons were associated with other axons, supports the idea that the sprouted axons tend to associate preferentially with other axons. Bilateral superior cervical ganglionectomies following cytochrome C infusion indicate that approximately 60% of the axons associated with the internal carotid artery arise from the superior cervical ganglion and that the majority of axons contacting the smooth muscle layer arise from this ganglion. Sympathectomy following NGF infusion resulted in a 79% reduction in the total number of perivascular axons, demonstrating overwhelmingly that the majority of sprouted axons are sympathetic fibers. These results demonstrate that infusion of NGF into the mature rat brain results in the preferential sprouting of sympathetic axons associated with the internal carotid artery. These findings are consistent with the hypothesis that NGF normally plays a role in the regulation of autonomic cerebrovascular innervation in the adult animal and that mature, uninjured sympathetic neurons remain responsive to NGF.

Trichloroethylene affects learning and decreases myelin in the rat hippocampus.

The goal of the present study was to relate previously observed behavioral effects with changes in myelin in the hippocampus following exposure to the industrial solvent 1,1,2-trichloroethylene (TCE). Young adult rats exposed to TCE via their drinking water underwent tests to evaluate their ability to perform spatial navigational tasks, and their brains were examined for changes in myelin in the hippocampus. Exposure to an average daily load of 5.5 mg TCE first for 4 weeks and then to 8.5 mg for an additional 2 weeks (separated by a 2-week interval) resulted in an increased level of performance in spatial navigational tasks. Examination of the brains from these animals revealed a significant decrease in the amount of myelin in one layer of the hippocampus, the stratum lacunosum-moleculare. No increase in performance was observed in rats exposed to an average daily load of 5.5 mg TCE for 4 weeks only. A reduction in myelin was observed, however, in the stratum lacunosum-moleculare of these animals. This decrease was not as severe as that seen in the twice-exposed animals. The results of this study suggest that exposure to TCE results in a reduction of hippocampal myelin, and that this reduction may be related to the increased level of performance observed following a second exposure to TCE.

Intracerebral NGF infusion induces hyperinnervation of cerebral blood vessels.

The use of intracerebral NGF (nerve growth factor) infusions as a therapeutic tool to prevent the degeneration of cholinergic neurons in humans suffering from Alzheimer's disease has recently been suggested. In the present study, intracerebroventricular infusion of nerve growth factor into the adult rat brain was found to induce axonal sprouting of mature, uninjured axons associated with the intradural segment of the internal carotid artery. Following NGF infusion, a three-fold increase in the total number of axons associated with the vessel wall was observed when compared with vehicle-infused animals. This vascular hyperinnervation might also occur in humans. Before NGF infusion therapy is initiated, more research is necessary concerning the specificity, mechanisms, and functional significance of the sprouting response observed in this study.

Maternal exposure to 1,1,2-trichloroethylene affects myelin in the hippocampal formation of the developing rat.

The effect of 1,1,2-trichloroethylene (TCE), an industrial solvent, on myelin in the dorsal hippocampus of the developing rat was investigated. Rat pups were exposed to TCE via their dams' drinking water while in utero and until they were sacrificed at 21 days of age. Frozen coronal sections through the dorsal hippocampus were stained for the presence of myelin using a modification of the Heidenhain procedure developed for frozen sections. A significant decrease in myelinated fibers was found in the stratum lacunosum-moleculare, an area comprised of distal dendritic profiles of CA1 pyramidal neurons which receive input from the entorhinal cortex. These findings suggest that the reduction in myelin in the hippocampus may be responsible, in part, for behavioral effects observed following TCE exposure.

Cholinergic innervation of canine thalamostriatal projection neurons: an ultrastructural study combining choline acetyltransferase immunocytochemistry and WGA-HRP retrograde labeling.

Choline acetyltransferase (ChAT) immunocytochemistry and lectin-conjugated horseradish peroxidase (WGA-HRP) histochemistry were combined at the electron microscopic level to examine the morphology of cholinergic terminals in the canine centrum medianum-parafascicular complex (CM-Pf) and to localize cholinergic terminals making synaptic contact with retrogradely labeled CM-Pf thalamostriatal projection neurons. Following WGA-HRP injections into the caudate nucleus, CM-Pf neurons were heavily labeled with WGA-HRP reaction product. Examination with the electron microscope revealed retrogradely labeled neurons characterized by a large nucleus with deep infoldings of the nuclear envelope. ChAT-positive terminals were observed arising from small-diameter nonmyelinated axonal profiles. These terminals varied in size from 0.5 to 1.4 micron in long diameter. The smaller terminals (0.5-0.7 micron) were seen most frequently and established symmetrical or slightly asymmetrical synaptic contacts with small dendritic profiles. The larger ChAT-positive terminals (1.0-1.4 micron) were less frequently observed, contained several mitochondria and small clusters of pleomorphic vesicles, and contacted large dendritic shafts and cell somata. Some of the postsynaptic targets of both smaller and larger ChAT-positive terminals were identified as belonging to retrogradely HRP-labeled thalamostriatal neurons. These observations indicate that at least some thalamostriatal neurons within the CM-Pf complex are innervated by cholinergic terminals which probably arise from ChAT-positive cell bodies located within the pontomesencephalic tegmentum, particularly within the nucleus tegmenti pedunculopontinus and the laterodorsal tegmental nucleus. These findings provide evidence for direct influence by cholinergic brainstem nuclei over activities of thalamostriatal neurons.