Adult Superior Cervical Ganglion Research Articles

Presence of Functional Neurotrophin TrkB Receptors in the Rat Superior Cervical Ganglion

Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc), but are not thought to express a functional full-length TrkB receptor (TrkB-Fl). We, and others, have demonstrated that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG) of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR), 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase) signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity.

Sympathetic reinnervation of peripheral targets following bilateral axotomy of the adult superior cervical ganglion

The ability of adult injured postganglionic axons to reinnervate cerebrovascular targets is unknown, yet these axons can influence cerebral blood flow, particularly during REM sleep. The objective of the present study was to assess quantitatively the sympathetic reinnervation of vascular as well as non-vascular targets following bilateral axotomy of the superior cervical ganglion (SCG) at short term (1 day, 7 day) and long term (8 weeks, 12 weeks) survival time points. The sympathetic innervation of representative extracerebral blood vessels [internal carotid artery (ICA), basilar artery (BA), middle cerebral artery (MCA)], the submandibular gland (SMG), and pineal gland was quantified following injury using an antibody to tyrosine hydroxylase (TH). Changes in TH innervation were related to TH protein content in the SCG. At 7 day following bilateral SCG axotomy, all targets were significantly depleted of TH innervation, and the exact site on the BA where SCG input was lost could be discerned. Complete sympathetic reinnervation of the ICA was observed at long term survival times, yet TH innervation of other vascular targets showed significant decreases even at 12 weeks following axotomy. The SMG was fully reinnervated by 12 weeks, yet TH innervation of the pineal gland remained significantly decreased. TH protein in the SCG was significantly decreased at both short term and long term time points and showed little evidence of recovery. Our data demonstrate a slow reinnervation of most vascular targets following axotomy of the SCG with only minimal recovery of TH protein in the SCG at 12 weeks following injury.

NGF protects paravertebral but not prevertebral sympathetic neurons against exposure to high glucose in vitro

In diabetes, sympathetic neuroaxonal dystrophy occurs in prevertebral celiac/superior mesenteric ganglia (CG/SMG) but not in paravertebral superior cervical ganglia (SCG). Changes in neurotrophic support by NGF occur during postnatal development and are implicated in diabetic neuropathy. Therefore, our aim was to compare the effects of age and NGF on the responses of CG/SMG and SCG neurons to high glucose levels in vitro. Neurons were dissociated from neonatal (5 days) and adult (12 weeks) rat ganglia and maintained in serum-free media containing glucose (10–100 mM) in the presence or absence of NGF (50 ng/ml) for 48 h. Cultures were immunostained for the pan neuronal marker, PGP9.5, and TUNEL. Neurons were assessed for viability, the presence of neurite outgrowth and for TUNEL-positive nuclei as a marker of apoptosis. Glucose caused significant concentration-dependent decreases in both viability and the proportion of neurons developing neurites together with significant increases in TUNEL-positive staining. Neonatal SCG neurons with neurites were significantly more susceptible to high glucose than adult SCG neurons whereas postnatal age had no influence on the response of CG/SMG neurons to high glucose. NGF protected adult SCG but not adult CG/SMG neurite-bearing neurons against the induction of TUNEL staining by high glucose. In the presence of NGF, CG/SMG neurons were markedly more susceptible to high glucose than SCG neurons. The greater susceptibility of CG/SMG neurons to diabetic neuropathy may be due to a selective inability of NGF to protect this particular population of sympathetic neurons against hyperglycaemia.

Norepinephrine transport-mediated gene expression in noradrenergic neurogenesis

BackgroundWe have identified a differential gene expression profile in neural crest stem cells that is due to deletion of the norepinephrine transporter (NET) gene. NET is the target of psychotropic substances, such as tricyclic antidepressants and the drug of abuse, cocaine. NET mutations have been implicated in depression, anxiety, orthostatic intolerance and attention deficit hyperactivity disorder (ADHD). NET function in adult noradrenergic neurons of the peripheral and central nervous systems is to internalize norepinephrine from the synaptic cleft. By contrast, during embryogenesis norepinephrine (NE) transport promotes differentiation of neural crest stem cells and locus ceruleus progenitors into noradrenergic neurons, whereas NET inhibitors block noradrenergic differentiation. While the structure of NET und the regulation of NET function are well described, little is known about downstream target genes of norepinephrine (NE) transport.ResultsWe have prepared gene expression profiles of in vitro differentiating wild type and norepinephrine transporter-deficient (NETKO) mouse neural crest cells using long serial analysis of gene expression (LongSAGE). Comparison analyses have identified a number of important differentially expressed genes, including genes relevant to neural crest formation, noradrenergic neuron differentiation and the phenotype of NETKO mice. Examples of differentially expressed genes that affect noradrenergic cell differentiation include genes in the bone morphogenetic protein (BMP) signaling pathway, the Phox2b binding partner Tlx2, the ubiquitin ligase Praja2, and the inhibitor of Notch signaling, Numbl. Differentially expressed genes that are likely to contribute to the NETKO phenotype include dopamine-β-hydroxylase (Dbh), tyrosine hydroxylase (Th), the peptide transmitter 'cocaine and amphetamine regulated transcript' (Cart), and the serotonin receptor subunit Htr3a. Real-time PCR confirmed differential expression of key genes not only in neural crest cells, but also in the adult superior cervical ganglion and locus ceruleus. In addition to known genes we have identified novel differentially expressed genes and thus provide a valuable database for future studies.ConclusionLoss of NET function during embryonic development in the mouse deregulates signaling pathways that are critically involved in neural crest formation and noradrenergic cell differentiation. The data further suggest deregulation of signaling pathways in the development and/or function of the NET-deficient peripheral, central and enteric nervous systems.

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.

Analysis of the Retrograde Transport of Glial Cell Line-Derived Neurotrophic Factor (GDNF), Neurturin, and Persephin Suggests ThatIn VivoSignaling for the GDNF Family is GFRα Coreceptor-Specific

Neurturin (NRTN) and glial cell line-derived neurotrophic factor (GDNF) are members of a family of trophic factors with similar actions in vitro on certain neuronal classes. Retrograde transport of GDNF and NRTN was compared in peripheral sensory, sympathetic, and motor neurons to determine whether in vivo these factors are transported selectively by different neuronal populations. After sciatic nerve injections, NRTN was transported by sensory neurons of the dorsal root ganglion (DRG). Competition studies demonstrated only limited cross-competition between NRTN and GDNF, indicating selective receptor-mediated transport of these factors. By using immunohistochemistry, we identified two populations of NRTN-transporting DRG neurons: a major population of small, RET-positive, IB4-positive, non-TrkA-expressing neurons that also show the ability to transport GDNF and a minor population of calretinin-expressing neurons that fail to transport GDNF. Spinal motor neurons in the adult showed relatively less ability to transport NRTN than to transport GDNF, although NRTN prevented the cell death of neonatal motor neurons in a manner very similar to GDNF (Yan et al., 1995) and persephin (PSPN) (Milbrandt et al., 1998). Last, NRTN, like GDNF, was not transported to sympathetic neurons of the adult superior cervical ganglion (SCG) after injection into the anterior eye chamber. These data reveal a high degree of functional selectivity of GDNF family receptor-alpha (GFRalpha) coreceptor subtypes for NRTN and GDNF in vivo.

Mitogen activated protein (MAP) kinase activity is increased in adult mouse superior cervical ganglia during culturing.

We have used adult mouse superior cervical ganglia (SCG) to study the role of mitogen activated protein (MAP) kinase activity during axonal outgrowth in vitro. An initial peak in activity within the first hours of culture was followed by a substantially higher activity after 1 to 2 days, a time when axons were actively growing. The latter peak is probably a result of both higher levels of protein and increased activity. The addition of nerve growth factor stimulated both outgrowth and kinase activity, whereas treating the cultures with the kinase inhibitor PD98059 had an opposite effect. Taken together, the results suggest that activation of the MAP kinase pathway could be involved in the initiation as well as regulation of axonal outgrowth from adult SCG.

Involvement of leukemia inhibitory factor in the increases in galanin and vasoactive intestinal peptide mRNA and the decreases in neuropeptide Y and tyrosine hydroxylase mRNA in sympathetic neurons after axotomy.

In response to axonal injury, noradrenergic sympathetic neurons of the adult superior cervical ganglion (SCG) alter their neurotransmitter phenotype. These alterations include increases in the levels of the neuropeptides, galanin, vasoactive intestinal peptide (VIP), and substance P (SP) and a decrease in the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH). Previous studies have indicated that after axotomy in vivo, leukemia inhibitory factor (LIF) plays an important role in increasing the contents of galanin and VIP in the SCG. In the present study, by examining the time courses of the changes in LIF and neuropeptide mRNA and by using LIF null mutant mice, we have determined that LIF alters neuropeptide content in part by increasing levels of peptide mRNA. In addition, LIF also makes a small contribution to the axotomy-induced down-regulation of mRNA encoding TH and neuropeptide Y, both of which are normally expressed at high levels in the SCG. Finally, by using a LIF-blocking antiserum, this cytokine was found to regulate SP expression in an in vitro axonal injury model. Thus, after axotomy, a single factor, LIF, participates in the down-regulation of peptides/proteins involved in normal neurotransmission and the up-regulation of a group of neuropeptides normally not present in the SCG that may be involved in regeneration.

A comparison between the adult rat and neonate rat of the architecture of sympathetic preganglionic neurones projecting to the superior cervical ganglion, stellate ganglion and adrenal medulla

Sympathetic preganglionic neurones (SPN) projecting to the superior cervical ganglion (SCG) and adrenal medulla (AM) in the neonate (< 14 days) and SCG, stellate ganglion (SG) and AM in the adult rat (> 3 months) were retrogradely labelled with cholera B horseradish peroxidase (CBHRP). Labelled neurones were found in 4 four distinct nuclei: the nucleus intermediolateralis thoracolumbalis pars principalis (ILp), a nucleus equivalent to the intemediolateral cell column (IML); the nucleus intermediolateralis thoracolumbalis pars funicularis (ILf); the nucleus intercalatus spinalis (IC) and the nucleus intercalatus pars paraependymatis (ICpe) or central autonomic area (CA). These were represented to a similar extent in both neonate and adult. Neonate and adult SCG, SG and AM-SPN had a similar segmental distribution cervical 8 (C8) to thoracic 5 (T5) for SCG-SPN and thoracic 3 (T3) to thoracic (T13) for AM-SPN whereas adult SG-SPN were distributed over segments C8 to T9. Most labelled neurones (70%) were located in the ILp with one segment containing the highest proportion of SPN. Three morphologically distinct neurones were evident. Fusiform and roundbodied were the most common. Fusiform somata of the ILp were orientated both mediolaterally and rostrocaudally in the neonate but only rostrocaudally in the adult. Dendrites of the SPN in the adult and neonate extended in a dense rostrocaudal band along the ILp, more diffusely into the white matter of the ILf and in bundles medially towards the central canal (CC). The neonate showed some significant differences. In the ILp, the cell bodies were less tightly packed into a narrow band and into clusters and the dendrites were more diffuse. It was concluded that at 12 days postnatally the organisation of the sympathetic nuclei had still nor reached the adult form. However, there is no extensive realignment of dendrites in the adult so the ILp remains an ‘open’ nucleus like the neonate.

Transient expression of somatostatin peptide is a widespread feature of developing sensory and sympathetic neurons in the embryonic rat.

Previous studies from this and other laboratories demonstrated that many embryonic sensory ganglion cells in the rat transiently express the catecholamine synthesizing enzyme tyrosine hydroxylase (TH), a trait not expressed by most mature sensory neurons. We, therefore, sought to determine whether transient expression was uniquely associated with catecholaminergic traits, or, alternatively, whether embryonic ganglion cells transiently expressed peptidergic properties as well. Of the four peptides examined (somatostatin [somatotropin release inhibiting factor] (SRIF), galanin (Gal), calcitonin gene-related peptide (CGRP), and substance P (SP)), only SRIF was found to be transiently expressed during early stages of sensory gangliogenesis. Surprisingly, SRIF immunoreactivity was observed in virtually all cranial and spinal sensory ganglion cells on embryonic day (E) 12.5. In addition to perikaryal labeling, intense SRIF immunoreactivity was also observed in the central and peripheral processes of E12.5 sensory neurons, suggesting the peptide may be released from nerve endings. The time course of SRIF appearance in cranial ganglion cells paralleled that previously described for TH, and double-labeling studies revealed extensive co-localization of these two phenotypes. By E16.5, however, the number of neurons expressing SRIF had diminished markedly, indicating that SRIF is only transiently expressed by most sensory neurons during early stages of ganglion development. An unexpected finding was that transient expression of SRIF is also a prominent feature of sympathetic ganglion cells; however, the temporal pattern of staining in the sympathetic and sensory ganglia differed substantially. Whereas virtually no SRIF staining was observed in E12.5 sympathetics, the vast majority of cells in the E16.5 superior cervical ganglion (SCG) were labeled. This contrasted sharply with the adult SCG, in which only low levels of SRIF expression were found. These findings demonstrate that SRIF peptide is transiently expressed at high levels in peripheral sensory and sympathetic neurons during embryogenesis. The time course and widespread distribution of SRIF expression indicates that the peptide may play a role in early stages of ganglion cell growth and development. Moreover, these data, in conjunction with previous studies demonstrating SRIF immunoreactivity in developing central neurons, suggest that transient expression of this peptide is a common property of diverse neuronal cell types.

Development of excitable membrane properties in mammalian sympathetic neurons

Using the whole-cell patch-clamp recording technique, resting membrane potentials (RPs), action potential (AP) waveforms, and the properties of voltage-activated inward Na+ (lNa) and Ca2+ (lCa) currents and outward K+ (lA,lK) currents were examined in embryonic and neonatal rat superior cervical ganglion (SCG) neurons as a function of time during development in vivo and in vitro. The passive and active membrane properties of neonatal SCG cells examined less than or equal to 24 hr after isolation were similar to those described previously for adult SCG neurons and for neonatal SCG cells maintained for several weeks in culture. Since recordings were obtained within hours of cell dissociations, it is assumed that the results reflect the membrane properties of neonatal SCG neurons in vivo at the time of isolation. When neonatal cells were examined as a function of time (up to approximately 2-3 weeks) in vitro, neither RPs nor AP waveforms varied measurably. Although absolute (inward and outward) current amplitudes increased in cells maintained in vitro, in parallel with increases in cell size, no changes in the time- or voltage-dependent properties of the currents were observed. Similar results were obtained for cells isolated on or after embryonic day 18.5 (greater than or equal to E 18.5). The membrane properties of E 14.5-16.5 SCG cells examined less than or equal to 24 hr after isolation, in contrast, were significantly different: mean lCa density was higher, and APs were broader than in greater than or equal to E 18.5 cells, and, in addition, lA was absent in these cells. When E 14.5-16.5 cells were examined after approximately 1 week in vitro, lCa densities and AP waveforms were indistinguishable from those in greater than or equal to E 18.5 SCG neurons, and lA was present. These studies reveal that rat SCG neurons are electrophysiologically mature early in development. Even lA, which seems to be the last voltage-gated current to develop, appears well before birth. As the appearance of lA correlates with decreased membrane excitability and AP shortening, it seems likely that the development of lA either reflects or regulates a marked change in the overall input and output properties of developing sympathetic neurons.

Biosynthesis, development, and regulation of neuropeptide Y in superior cervical ganglion culture.

The biosynthesis of neuropeptide Y (NPY) and norepinephrine (NE) has been examined in dissociated neuronal cultures from newborn rat superior cervical ganglion (SCG). NPY synthetic rate was measured by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis after incubation in medium containing a labeled amino acid. The authenticity of the NPY was confirmed by reverse-phase HPLC analyses of tryptic peptides. The NPY synthetic rate in cultures grown in complete serum free medium increased 30-fold after plating, in parallel to catecholamine synthesis; both NPY and the catecholamines reached the rate for adult SCG neurons. This development in culture is seen without spinal cord input, target organs, or significant numbers of glial cells. NPY synthesis was maintained in the face of a major decrease in the rate of NE production after cholinergic induction.

Differences in the effects of membrane depolarization on levels of preprosomatostatin mRNA and tyrosine hydroxylase mRNA in rat sympathetic neurons in vivo and in culture

Regulation of preprosomatostatin mRNA and tyrosine hydroxylase mRNA were examined in sympathetic neurons of the rat superior cervical ganglion (SCG). Surgical denervation of the adult SCG increased ganglion levels of preprosomatostatin (SS) mRNA more than 11-fold, and levels of the mRNA remained elevated 14 days after surgery. By contrast, denervation decreased levels of tyrosine hydroxylase (TH) mRNA. Potassium- or veratridine-induced membrane depolarization of cultured neonatal sympathetic neurons decreased levels of SS mRNA but elevated levels of TH mRNA. Sodium channel blockade with tetrodotoxin prevented the effects of veratridine on SS and TH mRNAs. In toto these observations suggest that transsynaptic nerve impulse activity and sympathetic neurons membrane depolarization decrease SS synthesis but increase TH synthesis at the mRNA level. Thus nerve impulse activity may alter the relative levels of different transmitters co-expressed in the same neuronal population by inhibiting levels of some species of mRNA while simultaneously stimulating levels of others.

Membrane currents in adult rat superior cervical ganglia in dissociated tissue culture

Intracellular and whole-cell recordings were made from young adult superior cervical ganglion neurones maintained in dissociated tissue culture. The neurones possessed an M-current, A-current and calcium-activated potassium currents in addition to inward sodium and calcium currents. The properties of these currents closely resembled those observed in intact sympathetic ganglia. In contrast to neurones cultured from foetal tissue, these adult cultures exhibited consistent responses to nicotinic agonists.

Synaptic functions in rat sympathetic neurons in microcultures. IV. Nonadrenergic excitation of cardiac myocytes and the variety of multiple-transmitter states

In the first 3 papers of this series (Furshpan et al., 1986a, b; Potter et al., 1986), a sensitive microculture procedure was used to show that sympathetic principal neurons, dissociated from newborn or adult superior cervical ganglia and grown singly on cardiac myocytes, display adrenergic, cholinergic, and purinergic functions, sometimes in isolation but more often in combination. In this paper we describe additional effects on cardiac myocytes evoked by these neurons; the effects were excitatory and insensitive to adrenergic blocking agents (and to agents that block the inhibitory effects of acetylcholine and purines). In some of these microcultures, evidence consistent with secretion of serotonin was obtained; the nonadrenergic excitatory effect was diminished or abolished by serotonin blockers or reserpine. Further evidence for serotonergic transmission is presented in the accompanying paper by Sah and Matsumoto (1987). In other cases, an as-yet-unidentified agent "X" also produced a nonadrenergic excitation. The X effect characteristically required a prolonged train of neuronal impulses, had a time course of 50-200 sec, and was insensitive to agents that affected the other transmitters, including serotonin. In addition, we discuss 2 remarkable features of the transmitter repertoire of the microcultured sympathetic neurons: expression of the several transmitters in a variety of combinations, including at-least-quadruple function, and expression of the transmitters within a particular combination in varying relative strengths. The result is a diversity of transmitter release greater than that previously reported for vertebrate or invertebrate neurons.

Synaptic functions in rat sympathetic neurons in microcultures. II. Adrenergic/cholinergic dual status and plasticity

This is the second in a series of papers that describes the use of a sensitive microculture procedure to investigate the transmitter status of sympathetic neurons. Cultured immature principal neurons, dissociated from the superior cervical ganglia of newborn rats, are known to be plastic with respect to transmitter status; under certain culture conditions, populations of neurons that display (at least) adrenergic properties at the outset can be induced to display a variety of cholinergic properties, including the formation of functional neuron-neuron cholinergic synapses, as adrenergic properties decline. With the microculture procedure described in the preceding paper (Furshpan et al., 1986a), we have examined the transmitter status of individual neonate-derived neurons during this transition. Many such neurons secreted both norepinephrine and ACh (adrenergic/cholinergic dual function); examination of such neurons with the EM revealed a mixed population of synaptic vesicles. Direct evidence for a transition via this dual status was obtained by serial physiological assays of 14 neurons. The neonate-derived neurons were markedly heterogeneous in the rate of change of transmitter status. Principal neurons derived from adult superior cervical ganglia also displayed dual status, but the incidence was lower than in neonate-derived neurons cultured for similar periods. In preliminary serial assays of adult-derived neurons, many of the neurons did not acquire detectable cholinergic function, but in two cases evidence consistent with plasticity was obtained. While it is known that several types of neurons will form functional junctions in the presence of agents that block electrical activity, sympathetic principal neurons have apparently not been tested. In microculture, neuron-neuron synapses and junctions with cardiac myocytes were formed by sympathetic neurons grown chronically in the presence of blocking concentrations of TTX and hexamethonium.

Anomalous migration of central nervous tissue to transplanted autonomic ganglia.

When fragments of adult superior cervical ganglion (SCG) were transplanted to the undamaged surface of the cerebellum of 6–14-day-old rats, a series of remarkable changes ensued that were apparent during the postoperative periods of three weeks to two years: external granule cells were either arrested at the brain surface in the form of ectopic laminae, or migrated anomalously out of the brain to invade the SCG graft. Not only were granule cells so affected, but other interneurons, such as basket and stellate cells as well. The associated neuropil itself became displaced. Thus, mossy fibre boutons formed synaptic glomeruli with dendrites of arrested granule cells at the cerebellar surface. Reactive astrocytes, forming either thin parallel sheets or thick processes, and packed with filaments, mingled closely with the arrested neurons; typical Bergmann glia were not observed. Extensive bridges from the cerebellum to the SCG contained not only synaptically mature interneurons but also rows of parallel fibres, mossy fibres forming mini-glomeruli with solitary granule cells, and reactive astroglia. Portions of the Purkinje arbor extended into the tissue bridges and received synapses on elongate spines from co-migrating parallel fibres. Astroglial cells received synapse-like contacts on their somata and processes. Similar CG grafts induced tissue bridges, predominantly astroglial, to arise from the area postrema or dorsal medulla and enter the graft. Control transplantations tested the specificity of these cellular reactions. Other tissue grafts included muscle, salivary gland and normal or pre-degenerated sciatic nerve. Only direct allografts of sciatic nerve appeared to cause displacement of scattered granule cells. Non-biological grafts such as dacron, silicone and polystyrene, while causing mechanical deformation and inflammation, did not alter normal granule cell migration. The anomalously migrating neurons and neuropil, which seem to have by-passed certain patterns of cellular development, may respond positively to some diffusible tropic substances. The further determination of certain influences that may elicit these unique tissue-tissue interactions, suggested in the framework of this study to be components of neural regeneration or degeneration, could elucidate some aspects of neuronal interactions.

Induction and maintenance of free postsynaptic membrane thickenings in the adult superior cervical ganglion

The superior cervical ganglion (SCG) of adult rats was exposed to GABA, either by long lasting microapplication (implantation of glass bulbs for 1-24 days) or in short term experiments (external application up to 6 h). Autoradiography showed that [3H] GABA accumulated selectively in satellite cells. The GABA produced the following effects: (1) Specialized membrane thickenings--similar in fine structural appearance to those seen as postsynaptic membrane thickenings at Gray type I synapses--were formed at the extrasynaptic dendritic surface of principal ganglion cells. (2) Morphometry revealed that the surface to volume ratio of dendrites increased significantly corresponding to an enlargement of their extrasynaptic surface as a result of the formation of spine-like projections. (3) Electrophysiology confirmed that, at least after short term application, the action potentials induced by preganglionic stimulation were heavily suppressed. These results suggest that, in the course of depressed ganglionic activity, so-called free postsynaptic membrane thickenings are generated and maintained in the SCG of adult rats even in the absence of significant axonal degeneration. The discussion focuses on two points: (1) possible similarities between the conditions of neurons after denervation and under the influence of GABA; (2) a possible role of GABA and other substances with inhibitory action in synaptogenesis.

Brain tissue transplanted to the anterior chamber of the eye: 3. Substitution of lacking central noradrenaline input by host iris sympathetic fibers in the isolated cerebral cortex developed in oculo

Fetal parietal cerebral cortex was homologously transplanted to the anterior chambers of the eyes of adult rats. The transplants got vascularized, proliferated, as measured by in vivo stereoscopic inspections, and differentiated into brain tissue similar to cortex cerebri in situ and survived for long times, greater than 41/2 months. Fibers from the intact sympathetic adrenergic ground plexus of the iris were able to innervate the transplants in an organotypic way regarding fluorescence morphology, pattern of distribution of the nerve terminals and, to a certain extent, density of innervation, the only variable parameter being density of innervation. Thus, in unpretreated or MAO inhibited transplants only rather few to scattered terminals could be found, while after preincubation in 10(-5)M alpha-methyl-noradrenaline the number of visible terminals was normal or slightly less than normal, as compared to cortex cerebri in situ. When superior cervical ganglia (SCG) were transplanted together with fetal cortex tissue to sympathetically denervated eyes the ingrowth in the cortex tissue was similar to that after single cortex transplantation combined with 5 day old SCG, while a marked hyperinnervation was encountered when combined with adult SCG. It is concluded that the developing cortex cerebri, deprived of its normal CNS source of adrenergic nerves, is able to receive sympathetic adrenergic nerves from the iris in an organotypic way upon transplantation to the anterior chamber of the eye.