Axons of Passage and Inputs to Superior Cervical Ganglion in Rat.

Wheat germ agglutinin-horseradish peroxidase was injected into the entire (0.8 µL) or partial (rostral or caudal, 0.1-0.3 µL) superior cervical ganglion (SCG) of the rat (male Sprague-Dawley, N = 35) to examine the distribution of neurons in the middle (MCG) and inferior (ICG) cervical ganglion that send axons bypass the SCG. Whole-mounts of the SCG, cervical sympathetic trunk (CST), MCG, ICG, and sections of the brainstem and spinal cord were prepared. With entire SCG tracer injection, neurons were labeled evenly in the MCG (left: 258, right: 121), ICG (left: 848, right: 681), and CST (up to 770). Some neurons grouped in a single bulge just rostral to the MCG, which we termed as the "premiddle cervical ganglion" (pMCG). The left pMCG (120) is larger and has more neurons than the right pMCG (82). Centrally, neurons were labeled in lamina IX of cervical segments (C1: 18%, C2: 46%, C3: 33%, C4: 3%), intermediate zone of thoracic segments (T1: 31%, T2: 35%, T3: 27%, T4: 7%), and intermediate reticular nuclei (96%) and perifacial zone (4%) of brainstem. The rostral and caudal SCG injection selectively labeled neurons mainly in brainstem, C1-C2 and in T1-T2, respectively. Before projecting to their peripheral targets, many neurons in pMCG, MCG and ICG run rostrally within the CST rather than segmentally through the closest rami, from the level of SCG or above. Neurons in pMCG and MCG may have similar or complementary function and those in brainstem may be involved in the vestibulo-autonomic interaction. Anat Rec, 301:1906-1916, 2018. © 2018 Wiley Periodicals, Inc.

Mitotic motors coregulate microtubule patterns in axons and dendrites.

Microtubules are nearly uniformly oriented in the axons of vertebrate neurons but are non-uniformly oriented in their dendrites. Studies to date suggest a scenario for establishing these microtubule patterns whereby microtubules are transported into the axon and nascent dendrites with plus-ends-leading, and then additional microtubules of the opposite orientation are transported into the developing dendrites. Here, we used contemporary tools to confirm that depletion of kinesin-6 (also called CHO1/MKLP1 or kif23) from rat sympathetic neurons causes a reduction in the appearance of minus-end-distal microtubules in developing dendrites, which in turn causes them to assume an axon-like morphology. Interestingly, we observed a similar phenomenon when we depleted kinesin-12 (also called kif15 or HKLP2). Both motors are best known for their participation in mitosis in other cell types, and both are enriched in the cell body and dendrites of neurons. Unlike kinesin-12, which is present throughout the neuron, kinesin-6 is barely detectable in the axon. Accordingly, depletion of kinesin-6, unlike depletion of kinesin-12, has no effect on axonal branching or navigation. Interestingly, depletion of either motor results in faster growing axons with greater numbers of mobile microtubules. Based on these observations, we posit a model whereby these two motors generate forces that attenuate the transport of microtubules with plus-ends-leading from the cell body into the axon. Some of these microtubules are not only prevented from moving into the axon but are driven with minus-ends-leading into developing dendrites. In this manner, these so-called "mitotic" motors coregulate the microtubule patterns of axons and dendrites.

Lack of dystrophin in mdx mice modulates the expression of genes involved in neuron survival and differentiation.

Duchenne muscular dystrophy is an X-linked disease characterized by progressive and lethal muscular wasting. Dystrophic patients, however, are also afflicted by several neurological disorders, the importance of which is generally underestimated. As promising therapies for muscles are currently in clinical trial stages, with the potential to provide an increase in the lifespan of young patients, determination of the genetic and molecular aspects characterizing this complex disease is crucial in order to allow the development of therapeutic approaches specifically designed for the nervous system. In this study, differences in gene expression in the superior cervical ganglion of postnatal day (P)5, P10 and 6-7-week-old wild-type and genetically dystrophic mdx mice were evaluated by DNA microarray analysis. The main aim was to verify whether the lack of dystrophin affected the transcript levels of genes related to different aspects of neuron development and differentiation. Ontological analysis of more than 500 modulated genes showed significant differences in genetic class enrichment at each postnatal date. Upregulated genes mainly fell in the categories of vesicular trafficking, and cytoskeletal and synaptic organization, whereas downregulated genes were associated with axon development, growth factors, intracellular signal transduction, metabolic processes, gene expression regulation, synapse morphogenesis, and nicotinic receptor clustering. These data strongly suggest that the structural and functional alterations previously described in both the autonomic and central nervous systems of mdx mice with respect to wild-type mice and related to crucial aspects of neuron life (i.e. postnatal development, differentiation, and plasticity) result not only from protein post-translational modifications, but also from direct and/or indirect modulation of gene expression.

Detection of characteristic distributions of phospholipid head groups and fatty acids on neurite surface by time-of-flight secondary ion mass spectrometry.

Neurons have a large surface because of their long and thin neurites. This surface is composed of a lipid bilayer. Lipids have not been actively investigated so far because of some technical difficulties, although evidence from cell biology is emerging that lipids contain valuable information about their roles in the central nervous system. Recent progress in techniques, e.g., mass spectrometry, opens a new epoch of lipid research. We show herein the characteristic localization of phospholipid components in neurites by means of time-of-flight secondary ion mass spectrometry. We used explant cultures of mouse superior cervical ganglia, which are widely used by neurite investigation research. In a positive-ion detection mode, phospholipid head group molecules were predominantly detected. The ions of m/z 206.1 [phosphocholine, a common component of phosphatidylcholine (PC) and sphingomyelin (SM)] were evenly distributed throughout the neurites, whereas the ions of m/z 224.1, 246.1 (glycerophosphocholine, a part of PC, but not SM) showed relatively strong intensity on neurites adjacent to soma. In a negative-ion detection mode, fatty acids such as oleic and palmitic acids were mainly detected, showing high intensity on neurites adjacent to soma. Our results suggest that lipid components on the neuritic surface show characteristic distributions depending on neurite region.

Chemical coding for cardiovascular sympathetic preganglionic neurons in rats.

Cocaine and amphetamine-regulated transcript peptide (CART) is present in a subset of sympathetic preganglionic neurons in the rat. We examined the distribution of CART-immunoreactive terminals in rat stellate and superior cervical ganglia and adrenal gland and found that they surround neuropeptide Y-immunoreactive postganglionic neurons and noradrenergic chromaffin cells. The targets of CART-immunoreactive preganglionic neurons in the stellate and superior cervical ganglia were shown to be vasoconstrictor neurons supplying muscle and skin and cardiac-projecting postganglionic neurons: they did not target non-vasoconstrictor neurons innervating salivary glands, piloerector muscle, brown fat, or adrenergic chromaffin cells. Transneuronal tracing using pseudorabies virus demonstrated that many, but not all, preganglionic neurons in the vasoconstrictor pathway to forelimb skeletal muscle were CART immunoreactive. Similarly, analysis with the confocal microscope confirmed that 70% of boutons in contact with vasoconstrictor ganglion cells contained CART, whereas 30% did not. Finally, we show that CART-immunoreactive cells represented 69% of the preganglionic neuron population expressing c-Fos after systemic hypoxia. We conclude that CART is present in most, although not all, cardiovascular preganglionic neurons but not thoracic preganglionic neurons with non-cardiovascular targets. We suggest that CART immunoreactivity may identify the postulated "accessory" preganglionic neurons, whose actions may amplify vasomotor ganglionic transmission.

Enhanced dense core granule function and adrenal hypersecretion in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a progressive developmental disorder resulting from loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2), a transcription regulatory protein. The RTT phenotype is complex and includes severe cardiorespiratory abnormalities, dysautonomia and behavioral symptoms of elevated stress. These findings have been attributed to an apparent hyperactivity of the sympathetic nervous system due to defects in brainstem development; however, the possibility that the peripheral sympathoadrenal axis itself is abnormal has not been explored. The present study demonstrates that the adrenal medulla and sympathetic ganglia of Mecp2 null mice exhibit markedly reduced catecholamine content compared with wild-type controls. Despite this, null animals exhibit significantly higher plasma epinephrine levels, suggesting enhanced secretory granule function in adrenal chromaffin cells. Indeed, we find that Mecp2 null chromaffin cells exhibit a cell autonomous hypersecretory phenotype characterized by significant increases in the speed and size of individual secretory granule fusion events in response to electrical stimulation. These findings appear to indicate accelerated formation and enhanced dilation of the secretory granule fusion pore, resulting in elevated catecholamine release. Our data therefore highlight abnormal catecholamine function in the sympathoadrenal axis as a potential source of autonomic dysfunction in RTT. These findings may help to explain the apparent 'overactivity' of the sympathetic nervous system reported in patients with RTT.

[Patterns of lipofuscin accumulation in ganglionic nerve cells of superior cervical ganglion in humans].

BACKGROUND/AIM: Considering available literature lipofuscin is a classical age pigment of postmitotic cells, and a consistently recognized phenomenon in humans and animals. Lipofuscin accumulation is characteristic for nerve cells that are postmitotic. This research was focused on lipofuscin accumulation in ganglionic cells (GC) (postganglionic sympathetic cell bodies) of superior cervical ganglion in humans during ageing. METHODS: We analysed 30 ganglions from cadavers ranging from 20 to over 80 years of age. As material the tissue samples were used from the middle portion of the ganglion, which was separated from the surrounding tissue by the method of macrodissection. The tissue samples were routinely fixed in 10% neutral formalin and embedded in paraffin for classical histological analysis, then three consecutive (successive) sections 5 microm thick were made and stained with hematoxylin and eosin method (HE), silver impregnation technique by Masson Fontana and trichrome stain by Florantin. RESULTS: Immersion microscopy was used to analyse patterns of lipofuscin accumulation during ageing making possible to distinguish diffuse type (lipofuscin granules were irregularly distributed and non-confluent), unipolar type (lipofuscin granules were grouped at the end of the cell), bipolar type (lipofuscin granules were concentrated at the two opposite ends of a cell with the nucleus in between at the center of a cell), annular type (lipofuscin granules were in the shape of a complete or incomplete ring around the nucleus) and a cell completely filled with lipofuscin (two subtypes distinguishing, one with visible a nucleus, and the other with invisible one). Even at the age of 20 there were cells with lipofuscin granules accumulated in diffuse way, but in smaller numbers; the GC without lipofuscin were dominant. Growing older, especially above 60 years, all of the above mentioned patterns of lipofuscin accumulation were present with the evident increase in cells completely filled with lipofuscin, but cells without lipofuscin were also present even in the oldest persons. CONCLUSION: Lipofuscin is present in all periods of ageing with a different intensity of accumulation. GC without the pigment, diffusely distributed, as well as very rare cells with a unipolar type of lipofuscin distribution are characteristic for the age of 20-60 years. In the age above 60 years, except the cells without pigment and diffuse accumulation type, there are also bipolar and annular types and forms in which cells are completely filled with lipofuscin granules.

Serotonin type 1D receptors (5HTR) are differentially distributed in nerve fibres innervating craniofacial tissues.

We tested the hypothesis that the 5HT(1D)R, the primary antinociceptive target of triptans, is differentially distributed in tissues responsible for migraine pain. The density of 5HT(1D)R was quantified in tissues obtained from adult female rats with Western blot analysis. Receptor location was assessed with immunohistochemistry. The density of 5HT(1D)R was significantly greater in tissues known to produce migraine-like pain (i.e. circle of Willis and dura) than in structures in which triptans have no antinociceptive efficacy (i.e. temporalis muscle). 5HT(1D)R-like immunoreactivity was restricted to neuronal fibres, where it colocalized with calcitonin gene-related peptide and tyrosine hydroxylase immunoreactive fibres. These results are consistent with our hypothesis that the limited therapeutic profile of triptans could reflect its differential peripheral distribution and that the antinociceptive efficacy reflects inhibition of neuropeptide release from sensory afferents. An additional site of action at sympathetic efferents is also suggested.

PKA-catalyzed phosphorylation of tomosyn and its implication in Ca2+-dependent exocytosis of neurotransmitter.

Neurotransmitter is released from nerve terminals by Ca2+-dependent exocytosis through many steps. SNARE proteins are key components at the priming and fusion steps, and the priming step is modulated by cAMP-dependent protein kinase (PKA), which causes synaptic plasticity. We show that the SNARE regulatory protein tomosyn is directly phosphorylated by PKA, which reduces its interaction with syntaxin-1 (a component of SNAREs) and enhances the formation of the SNARE complex. Electrophysiological studies using cultured superior cervical ganglion (SCG) neurons revealed that this enhanced formation of the SNARE complex by the PKA-catalyzed phosphorylation of tomosyn increased the fusion-competent readily releasable pool of synaptic vesicles and, thereby, enhanced neurotransmitter release. This mechanism was indeed involved in the facilitation of neurotransmitter release that was induced by a potent biological mediator, the pituitary adenylate cyclase-activating polypeptide, in SCG neurons. We describe the roles and modes of action of PKA and tomosyn in Ca2+-dependent neurotransmitter release.

The origin of extrinsic nitrergic axons supplying the human eye.

Nitrergic nerve fibres of intrinsic and extrinsic origin constitute an important component of the autonomic innervation in the human eye. The intrinsic source of nitrergic nerves are the ganglion cells in choroid and ciliary muscle. In order to obtain more information on the origin of extrinsic nitrergic nerves in the human eye, we obtained superior cervical, ciliary, pterygopalatine and trigeminal ganglia from six human donors, and stained them for neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-D). In the superior cervical ganglia, nNOS/NADPH-D-positive varicose axons were observed whereas perikarya were consistently negative. Fewer than 1% of perikarya in the ciliary ganglia were labelled for nNOS/NADPH-D. The diameter of nNOS/NADPH-D-positive ciliary perikarya was between 8 and 10 microm, which was markedly smaller than the diameter of the vast majority of negative perikarya in the ciliary ganglion. More than 70% of perikarya in the pterygopalatine ganglia were intensely labelled for both nNOS and NADPH-D. In trigeminal ganglia, 18% of perikarya were nNOS/NADPH-D-positive. The average diameter of trigeminal nNOS/NADPH-D perikarya was between 25 and 45 microm. Pterygopalatine and trigeminal ganglia are the most likely sources for extrinsic nerve fibres to the human eye.

Caldesmon tethers myosin V to actin and facilitates in vitro motility.

The current study examines the hypothesis that caldesmon can facilitate the interaction of myosin V with actin filaments by tethering myosin V to actin. Myosin V, purified from bovine brain stem, translocated actin filaments in an in vitro motility assay at a velocity of 0.30+/-0.05 microm/s in the absence of caldesmon at a myosin concentration of 50 microg/ml (ionic strength=110 mM). Filament binding and motility was absent when the myosin concentration applied to the coverslip was reduced to 5 microg/ml, however, the addition of 0.4 microM caldesmon restored binding and motility (0.28+/-0.06 microm/s). This restoration of motility in the presence of caldesmon was blocked by an N-terminal fragment of caldesmon that competitively inhibits the binding of intact caldesmon to myosin. Similar to previous findings with both smooth muscle myosin and platelets (Haeberle et al., 1992; Hemric et al., 1994), these results demonstrate that caldesmon can form a mobile tether that maintains the proximity of myosin V with actin filaments without restricting filament sliding.

Zinc transporter 3 and zinc ions in the rodent superior cervical ganglion neurons.

Previous studies have revealed that zinc-enriched (ZEN) terminals are present in all parts of the CNS though with great differences in intensity. The densest populations of both ZEN terminals and ZEN somata are found in telencephalic structures, but also structures like the spinal cord demonstrate impressive ZEN systems spreading terminals several segments around the respective ZEN somata. The present study evaluates whether sympathetic neurons in the superior cervical ganglia (SCG) are ZEN neurons, i.e. contain vesicles that have zinc transporter 3 (ZnT3) proteins in their membranes and contain zinc ions. ZnT3 immunoreactivity (IR) was found in the somata and processes in the postganglionic neurons of mouse SCG. Only a small fraction of neurons (less than 5%), expressed varying degrees of ZnT3. Colchicine treatment, however, increased the number of ZnT3-positive neurons three-fold, suggesting an accumulation of ZnT3 protein in the somata. A small proportion of the postganglionic axons revealed dotted accumulations of ZnT3 IR along their courses. Double labeling showed that all ZnT3-positive neurons and axons were also tyrosine hydroxylase-positive with strong immunofluorescence, while no colocalization was found between ZnT3 and the vesicular acetylcholine transporter (VAChT) or neuropeptide Y IR. VAChT-positive preganglionic neurons were found to terminate on ZnT3 neuronal somata. 6-Methoxy 8-para toluene sulfonamide quinoline fluorescence and zinc selenium autometallography (ZnSe(AMG)) revealed that a subgroup of SCG cells contained free or loosely bound zinc ions. It is therefore concluded that ZnT3 and zinc ions are present in a subpopulation of TH-positive, NPY-negative neurons in the rodent SCG, supporting the notion that vesicular zinc ions may play a special role in the peripheral sympathetic adrenergic system.

Subtype-specific coupling with ADP-ribosyl cyclase of metabotropic glutamate receptors in retina, cervical superior ganglion and NG108-15 cells.

Cyclic ADP-ribose (cADP-ribose) is a putative second messenger or modulator. However, the role of cADP-ribose in the downstream signals of the metabotropic glutamate receptors (mGluRs) is unclear. Here, we show that glutamate stimulates ADP-ribosyl cyclase activity in rat or mouse crude membranes of retina via group III mGluRs or in superior cervical ganglion via group I mGluRs. The retina of mGluR6-deficient mice showed no increase in the ADP-ribosyl cyclase level in response to glutamate. GTP enhanced the initial rate of basal and glutamate-stimulated cyclase activity. GTP-gamma-S also stimulated basal activity. To determine whether the coupling mode of mGluRs to ADP-ribosyl cyclase is a feature common to individual cloned mGluRs, we expressed each mGluR subtype in NG108-15 neuroblastoma x glioma hybrid cells. The glutamate-induced stimulation of the cyclase occurs preferentially in NG108-15 cells over-expressing mGluRs1, 3, 5, and 6. Cells expressing mGluR2 or mGluRs4 and 7 exhibit inhibition or no coupling, respectively. Glutamate-induced activation or inhibition of the cyclase activity was eliminated after pre-treatment with cholera or pertussis toxin, respectively. Thus, the subtype-specific coupling of mGluRs to ADP-ribosyl cyclase via G proteins suggests that some glutamate-evoked neuronal functions are mediated by cADP-ribose.

Re-establishment of neurochemical coding of preganglionic neurons innervating transplanted targets.

We investigated the effect on neurochemical phenotype of changing the targets innervated by sympathetic preganglionic neurons. In neonatal rats, the adrenal gland was transplanted into the neck, to replace the postganglionic neurons of the superior cervical ganglion. Transplanted adrenal glands survived, and contained noradrenergic and adrenergic chromaffin cells, and adrenal ganglion cells. Retrograde tracing from the transplants showed that they were innervated by preganglionic neurons that would normally have supplied postganglionic neurons of the superior cervical ganglion. The neurochemical phenotypes of preganglionic axons innervating transplanted chromaffin cells were compared with those innervating the normal adrenal medulla or superior cervical ganglion neurons. As in the normal adrenal gland, preganglionic nerve fibres apposing transplanted chromaffin cells were cholinergic. The peptide and calcium-binding protein content of preganglionic fibres was similar in normal and transplanted adrenal glands. In both cases, cholinergic fibres immunoreactive for enkephalin targeted adrenergic chromaffin cells, whilst cholinergic fibres with co-localised calretinin-immunoreactivity innervated noradrenergic chromaffin cells and adrenal ganglion cells. In contrast to the innervation of normal adrenal glands, these axons lacked immunoreactivity to nitric oxide synthase. In a set of control experiments, the superior cervical ganglion was subjected to preganglionic denervation in rat pups the same age as those that received adrenal transplants, and the ganglion was allowed to be re-innervated over the same time course as the adrenal transplants were studied. When the superior cervical ganglion was re-innervated by preganglionic nerve fibres, we observed that all aspects of chemical coding were restored, including cholinergic markers, nitric oxide synthase, enkephalin, calcitonin gene-related peptide and calcium binding proteins in predicted combinations, although the density of nerve fibres was always lower in re-innervated ganglia. These data show that the neurochemical phenotypes expressed by preganglionic neurons re-innervating adrenal chromaffin cells are selective and similar to those seen in the normal adrenal gland. Two explanations are advanced: either that contact of preganglionic axons with novel target cells has induced a switch in their neurochemical phenotypes, or that there has been target-selective reinnervation by pre-existing fibres of appropriate phenotype. Regardless of which of these alternatives is correct, the restoration of normal preganglionic codes to the superior cervical ganglion following denervation supports the idea that the target tissue influences the neurochemistry of innervating preganglionic neurons.

A splice variant of the G protein beta 3-subunit implicated in disease states does not modulate ion channels.

A single-nucleotide polymorphism (C825T) in the GNB3 gene produces an alternative splice variant of the heterotrimeric G protein beta3 subunit (Gbeta3). Translation of the alternatively spliced mRNA results in a protein product, Gbeta3-s, in which 41 amino acids are deleted from Gbeta3. Interestingly, previous studies indicate that the C825T allele occurs with a high frequency in patients with certain vascular disorders. However, little information is available regarding the functional role Gbeta3-s might play in ion channel modulation. To examine this aspect, Gbeta3 or Gbeta3-s, along with either Ggamma2 or Ggamma5, were expressed in rat sympathetic neurons by nuclear microinjection of vector encoding the desired protein. In contrast to Gbeta3, expression of Gbeta3-s did not modulate N-type Ca(2+) or G protein-gated inwardly rectifying K(+) channels. In addition, Gbeta3-s did not appear to complex with a pertussis toxin-insensitive mutant of Galpha(i2) or couple to natively expressed alpha(2)-adrenergic receptors. Finally, fluorescence resonance energy transfer (FRET) measurements indicated that enhanced yellow fluorescent protein (EYFP)-labeled Gbeta3-s does not form a Gbetagamma heterodimer when coexpressed with enhanced cyan fluorescent protein (ECFP)-labeled Ggamma2. Therefore, when expressed in sympathetic neurons, Gbeta3-s appears to lack biological activity--hence pathological conditions in patients carrying the homozygous C825T allele may result from a functional knockout of Gbeta3.

A method utilizing differential culture and comparative RT-PCR for determining RNA expression in superior cervical ganglion neurones.

In order to assess the neuronal expression of caspase mRNA in primary cultures of rat superior cervical ganglion (SCG) neurones a method of differential cell purification and comparative RT-PCR was devised. SCG primary cultures generally contain variable percentages of non-neuronal contaminants, which influence RT-PCR results. We optimised a neuronal purification method, allowing the preparation of both highly purified neuronal cultures and mixed cultures, enriched in non-neuronal contaminants. These two sets of cells were cultured in parallel and subsequently analysed by RT-PCR. The use of cell type specific oligonucleotides allowed evaluation of the relative distribution of neuronal (neurofilament) and non-neuronal transcripts in the two cultures. In parallel, specific oligonucleotides were used to detect the mRNA levels of caspase family members. The partition of neurofilament transcript between pure and mixed cultures was found to be statistically different from the partition of the non-neuronal markers. Therefore statistical difference from the partition of non-neuronal markers was taken as evidence for expression in neurones. We show that caspase-2, -3, -6, -7 and -9 transcripts are expressed in SCG neurones whereas caspase-1 is probably absent. Furthermore, none of these transcripts are upregulated during neuronal death induced by nerve growth factor withdrawal. This method could be applied to the analysis of other transcripts in SCG and other primary neuronal cultures containing significant percentages of contaminant cell types.

Enhanced tissue plasminogen activator synthesis by the sympathetic neurons that innervate aging vessels.

We investigated the source of the increased release of tissue plasminogen activator (t-PA) into the circulation that occurs during natural aging. Both the basal release and the acute stress-associated release induced by sympathetic stimulations are greater in older subjects. It is widely assumed that the source of these increases is vascular endothelium. However, the sympathetic neurons that densely innervate resistance vessel walls were recently shown to synthesize and transport active t-PA to axon terminals in vascular smooth muscle, suggesting an alternative source. These fine t-PA-bearing axons lie in the seldom-studied deep adventitia of vessel walls, where they are less visible than endothelium in tissue sections. Using Northern blot analysis, we observed that t-PAmRNA synthesis is increased 54% in the ganglion parent neuron cell bodies that innervate aged vessels. The t-PA release from isolated, aged ganglia in cultures was twofold greater than that from younger controls. In addition, aged whole-artery explants showed a 20% greater basal and a 50% greater acute release of stored t-PA in vitro. In vivo levels of active t-PA were 33% greater in the blood and 40% greater in the aqueous humor. These results are consistent with an increased infusion of the active t-PA protease from sympathetic axon terminals into the vessel wall extracellular matrix and the blood during natural aging, in addition to the basal endothelial release. We suggest that the cumulative impact of an accelerated plasmin production and matrix degradation within vessel walls, especially during repetitive stress, may play an unrecognized role in the pathogenesis of vascular aging. The possibility that increased sympathetic nervous system plasminogenesis influences the aging process in nonvascular tissues also deserves further investigation.

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.

Differential composition of 5-hydroxytryptamine3 receptors synthesized in the rat CNS and peripheral nervous system.

The type 3 serotonin (5-HT3) receptor is the only ligand-gated ion channel receptor for serotonin in vertebrates. Two 5-HT3 receptor subunits have been cloned, subunit A (5-HT3A) and subunit B (5-HT3B). We used in situ hybridization histochemistry and reverse transcriptase-PCR amplification to demonstrate that 5-HT3A subunit transcripts are expressed in central and peripheral neurons. In contrast, 5-HT3B subunit transcripts are restricted to peripheral neurons. Thus, the prevalent form of 5-HT3 receptor synthesized within the CNS lacks the 5-HT3B subunit. Because coexpression of 5-HT3A and 5-HT3B subunits produces heteromeric 5-HT3A/3B receptors with properties that differ from those of 5-HT3A homomeric receptors, we investigated possible coexpression of both subunits at the cellular level. We found that near to 90% of all 5-HT3B expressing neurons coexpress the 5-HT3A subunit in superior cervical and nodose ganglia (NG). In addition, there is a cellular population that expresses only the 5-HT3A subunit. Therefore, peripheral neurons have the capacity to synthesize two different 5-HT3 receptors, 5-HT3A+/3B- and 5-HT3A+/3B+ receptors. We also determined that neurons of NG projecting to the nucleus tractus solitarium and those of dorsal root ganglia projecting to superficial layers of the spinal cord express 5-HT3A or 5-HT3A/3B subunits. Thus, presynaptic 5-HT3 receptors containing the 5-HT3B subunit might be present in these target brain areas. The compartmentalized structural composition of the 5-HT3 receptor may be the basis of functional diversity within this receptor. This raises the possibility that 5-HT3 receptors participating in sympathetic, parasympathetic and sensory functions may be functionally different from those involved in cognition and emotional behavior.

Dystrophin stabilizes alpha 3- but not alpha 7-containing nicotinic acetylcholine receptor subtypes at the postsynaptic apparatus in the mouse superior cervical ganglion.

The nicotinic acetylcholine receptor (nAChR) subtypes were characterized in the superior cervical ganglion (SCG) of wild-type and dystrophin-lacking mdx mice. The binding of Epibatidine and alphaBungarotoxin, ligands for alpha3- and alpha7-containing receptors, respectively, revealed, for each ligand, a single class of high-affinity binding sites, with similar affinity in both wild-type and mdx mice. The Epibatidine-labeled receptors were immunoprecipitated by antibodies against the alpha3, beta2, and beta4 subunits. Immunocytochemistry showed that the percentage of alpha3-, beta2-, and beta4- but not of alpha7-immunopositive postsynaptic specializations was significantly lower in mdx than in wild-type mouse SCG. These observations suggest that the mouse SCG contains nAChRs, stabilized by dystrophin, in which the alpha3 subunit is associated with the beta2 and/or beta4 subunits. Conversely, dystrophin is not involved in the stabilization of the alpha7-containing nAChRs, as the percentage of alpha7-immunopositive synapses is similar in both wild-type and mdx mouse SCG.