[Early clinical outcomes of minimally invasive coronary surgery-total arterial coronary revascularization].

Objective: To summarize the early experience and clinical value of minimally invasive coronary surgery-total arterial coronary revascularization (MICS-TACR) through the left anterior small incision. Methods: Between May 2015 and June 2018, a total of 31 consecutive cases [21 males and 10 females, aged (63.2±9.3) years] in Peking University Third Hospital who were performed MICS-TACR with bilateral internal mammary artery and radial artery were enrolled. Meanwhile, 1 489 cases of conventional median sternal incision off-pump coronary artery bypass grafting (OPCABG) were matched as control group. According to exclusion criteria and matching score, 90 cases [55 males and 35 females, aged (63.8±9.5) years] were selected as the control group, and the perioperative data of the two groups were compared. All patients in MICS-TACR group underwent postoperative angiography and the graft patency was evaluated. Results: There were no statistically significant differences in baseline data between the two groups. The perioperative blood transfusion of MICS-TACR group was less than control group [0(0,0) U vs 0(0,4) U, P=0.003]. There were no statistically significant differences in operative mortality, intraoperative and postoperative intra-aortic balloon pump (IABP) and extracorporeal membrane oxygenation (ECMO) use, re-operation rate, perioperative major adverse cardiac and cerebrovascular events (MACCE), new-onset renal failure, atrial fibrillation, and multiple organ failure between the two groups. Postoperative angiography showed that there was no significant difference in the patency rate between the MICS-TACR group and control group(all P>0.05). Conclusion: Total arterial coronary revascularization can be successfully accomplished under the left anterior small incision, and the early clinical outcome is satisfied.

[Comparison of outcomes of two minimally invasive approaches for multi-vessel coronary revascularization].

OBJECTIVE: To compare the safety and effectiveness of two minimally invasive approaches for multi-vessel coronary revascularization. METHODS: From August 2014 to February 2017, 70 consecutive patients who underwent minimally invasive coronary artery bypass grafting in Peking University Third Hospital were randomly divided into two groups. In one group, 40 patients underwent staged-hybrid coronary revascularization (staged-HCR) treatment; in the other group, 30 patients underwent minimally invasive total arterial revascularization with bilateral internal thoracic artery (BITA). In staged -HCR group, the patients underwent minimally invasive direct coronary artery bypass grafting (MIDCAB) and percutaneous coronary intervention (PCI) procedure for treatment of multi-vessel disease. In BITA group, the patients underwent total arterial coronary artery bypass grafting with composite "Y" BITA graft. Preoperative and postoperative data of the two groups, including postoperative blood usage, mechanical ventilation time, domiciling duration in intensive care unit (ICU), major adverse cerebral and cardiovascular event (MACCE), and postoperative coronary angiography results were compared, in order to evaluate the safety and effectiveness of these surgical approaches. RESULTS: The preoperative characteristics of 70 patients in the two groups showed no significant difference. All the patients underwent successfully, elective minimally invasive multi-vessel coronary artery bypass grafting as scheduled preoperatively. Postoperative result showed the patients in staged-HCR group took advantages in less postoperative mechanical ventilation time [Staged-HCR group (11.2±8.7) h vs. BITA group (18.3±9.1) h, P=0.013], shorter domiciling duration in ICU [Staged-HCR group (26.29±4.05) h vs. BITA group (44.74±28.75) h, P=0.022], and less total drainage [Staged-HCR group (695.57±250.46) mL vs. BITA group (1 103.26±547.44) mL, P=0.03] than the patients in the group of minimally invasive total arterial revascularization with BITA. Postoperative in hospital coronary angiography showed satisfactory graft patency rates in both groups [97.5% in Staged-HCR group vs. 97.8% in BITA group]. No MACCE occurred in both groups during hospitalization. CONCLUSION: Staged-HCR is a feasible method for the treatment of multi-vessel revascularization involving right coronary artery. Minimally coronary revascularization with BITA is associated with superior long-term graft patency and it's recommended for patients who could not tolerate dual-antiplatelet therapy. This study shows that both minimally invasive surgical approaches are safe and effective for treatment of patients with multi-vessel coronary artery disease.

[Effect of suture canalization on the treatment of primary open-angle glaucoma: 3 years clinical observation].

OBJECTIVE: To evaluate the safety and effectiveness of the Suture Canalization, which is a kind of Schlemm canal surgery, on the treatment of patients with primary open angle glaucoma (POAG). METHODS: Retrospective cases series study. Fifty-two patients (52 eyes) with POAG were recruited from October 2007 to July 2009 from Hospital of Optometry and Ophthalmology, Wenzhou Medical University. Twenty-six patients (26 eyes) were included in the study group, who were treated with the Suture Canalization surgery with trabeculectomy. Twenty-six patients (26 eyes) were included as the control group, who were treated with trabeculectomy only. Best correct visual acuity (BCVA), intraocular pressure (IOP), bleb morphology, postoperative treatment of medicine of anti-glaucoma and complications of surgery were recorded at 1 d, 1 week, 1, 3, 6, 12, 18, 24, 30 and 36 months after surgery. After testing the normality and homogeneity of variance of the data of the multiple sets of measurement data, the variance analysis was adopted, and the t test was used in the comparison of two groups. Counting data using chi-square test, the level of variables between the groups should be compared with the rank sum test. RESULTS: No significant difference was found in baseline data, as well as the changes of BCVA between the study group and control group at the last visit (χ(2)=3.06, P=0.08) . Complete and qualified success was achieved in 18 (69.2%) and 23 (88.5%) cases separately in study group, 16 (61.5%) and 22 (84.6%) cases separately in control group (χ(2)=0.17, P=0.69) . There was no significant difference between two groups. IOP was decreased post-operatively: from (32.4±9.3) mmHg(1 mmHg=0.133 kPa)to (16.9±3.5) mmHg at the last visit in study group and (31.3±10.0) mmHg to (15.5±4.6) mmHg at the last visit in control group. There were significant difference between last visit and preoperative IOP between two groups (study group: t=8.12, P<0.01; control group: t=7.20, P<0.01). No significant difference was found between two groups in the decreased amplitude of IOP with treatment at the last visit (t=0.23, P=0.63), as well as the postoperative IOP at the different visits (P>0.05). 65.4% of cases in the study group and 61.5% patients in control group formed functional blebs (diffuse type and microcapsule type) at the last visit. No significant difference between the IOP of patients with functional blebs (15.5±3.5) mmHg and those with non-functional blebs (16.0±3.4) mmHg in study group at the last visit (t=-0.49, P=0.64). No serious complications during and after surgery were found in subjects of the both groups. No significant difference was present in medicine amounts at last visit between the two groups (t=2.93, P=0.09). CONCLUSIONS: Suture Canalization is a safe and effective surgical procedure to decrease intraocular pressure in patients with POAG, through Schlemm canal and external filtration. While it maybe not suitable to the patient with high pre-operative IOP.(Chin J OPhthalmol, 2016, 52: 416-421).

Developmental changes in the expression of calbindin and potassium-channel subunits Kv3.1b and Kv3.2 in mouse Renshaw cells.

One class of spinal interneurons, the Renshaw cells, is able to discharge at very high frequencies in adult mammals. Neuronal firing at such high frequencies requires voltage-gated potassium channels to rapidly repolarize the membrane potential after each action potential. We sought to establish the pattern of expression of calbindin and potassium channels with Kv3.1b and Kv3.2 subunits in Renshaw cells at different developmental stages of postnatal mice. The pattern of expression of calbindin changed dramatically during early postnatal development. An adult pattern of calbindin reactive neurons started to emerge from postnatal day 10 to postnatal day 14, with cells in laminae I and II of superficial dorsal horn and the ventral lamina VII. Renshaw cells were identified immunohistochemically by their expression of calbindin and their location in the ventral horn of the spinal cord. Western blot results of the lumbar spinal cord showed that Kv3.1b expression became faintly evident from postnatal day 10, reached a maximum at postnatal day 21 and was maintained through postnatal day 49. Double labeling results showed that all Renshaw cells expressed Kv3.1b weakly from postnatal day 14, and strongly at postnatal day 21. Western blot results showed that Kv3.2 expression became detectable in the lumbar cord from postnatal day 12, and increased steadily until reaching an adult level at postnatal day 28. In contrast to the Kv3.1b results, Kv3.2 was not expressed in Renshaw cells, although some neurons located at laminae VIII and VI expressed Kv3.2. We conclude that Renshaw cells express Kv3.1b but not Kv3.2 from postnatal day 14.

alpha2a adrenoceptors regulate phosphorylation of microtubule-associated protein-2 in cultured cortical neurons.

Adrenoceptors have been suggested to mediate neuronal development. This study revealed the expression of alpha2A adrenoceptors in the cortical plate of fetal mouse cerebral wall. The effects of alpha2A adrenoceptor on dendrite growth were investigated in primary neuronal cultures. Application of alpha2 adrenoceptor agonists, BHT 933 or UK 14304 for 24 or 72 h resulted in a 1.5-2-fold increase in dendrite lengths. This effect was blocked by alpha2 adrenergic antagonists, RX 821002 or yohimbine, as well as a alpha2A selective antagonist, BRL 44408, but not by alpha2B/alpha2C selective antagonists ARC 239, imiloxan and rauwolscine. Guanfacine, a alpha2A selective agonists, also significantly increased the dendrite lengths in culture. These results suggest that the morphological effect is wholly attributable to alpha2A adrenoceptor activation. We further tested the hypothesis that alpha2A adrenoceptors act through altering the phosphorylation state of microtubule-associated protein 2. The results showed that the phosphorylation of microtubule-associated protein 2 was significantly reduced on both serine and threonine residues by over 40% after 2 h of application of guanfacine and was maintained at this low level for a prolonged time up to 96 h. These findings suggest that alpha2A adrenoceptors regulate the phosphorylation of microtubule-associated protein 2, which in turn mediates dendrite growth of cortical neurons.

D1 dopamine receptor regulation of microtubule-associated protein-2 phosphorylation in developing cerebral cortical neurons.

This study addresses the hypothesis that the previously described capacity of D1 dopamine receptors (D1Rs) to regulate dendritic growth in developing cortical neurons may involve alterations in the phosphorylation state of microtubule-associated protein-2 (MAP2). The changes in phosphorylation of this protein are known to affect its ability to stabilize the dendritic cytoskeleton. The study involved two systems: primary cultures of mouse cortical neurons grown in the presence of the D1R agonists, SKF82958 or A77636, and the cortex of neonatal transgenic mice overexpressing the D1A subtype of D1R. In both models, a decrease in dendritic extension corresponded with an elevation in MAP2 phosphorylation. This phosphorylation occurred on all three amino acid residues examined in this study: serine, threonine, and tyrosine. In cultured cortical neurons, D1R stimulation-induced increase in MAP2 phosphorylation was blocked by the protein kinase A (PKA) inhibitor, H-89, and mimicked by the PKA activator, S(p)-cAMPS. This indicates that D1Rs modulate MAP2 phosphorylation through PKA-associated intracellular signaling pathways. We also observed that the elevations in MAP2 phosphorylation in neuronal cultures in the presence of D1R agonists (or S(p)-cAMPS) were maintained for a prolonged time (up to at least 96 hr). Moreover, MAP2 phosphorylation underwent a substantial increase between 24 and 72 hr of exposure to these drugs. Our findings are consistent with the idea that D1Rs can modulate growth and maintenance of dendrites in developing cortical cells by regulating the phosphorylation of MAP2. In addition, our observations suggest that MAP2 phosphorylation by long-term activation of D1Rs (and PKA) can be divided into two phases: the initial approximately 24-hr-long phase of a relatively weak elevation in phosphorylation and the delayed phase of a much more robust phosphorylation increase taking place during the next approximately 48 hr.

Mechanism of measles virus failure to activate NF-kappaB in neuronal cells.

Lack of IFN-beta and MHC class I expression in measles virus (MV) infected neurons could impair the host antiviral defense mechanism and result in virus escape from recognition by cytotoxic T-cells. Induction of IFN-beta and MHC class I gene expression requires NF-kappaB activation which depends on degradation of IkappaBalpha, an inhibitory protein of NF-kappaB. In earlier studies we demonstrated that in contrast to glial cells, MV was unable to induce IkappaBalpha degradation in neuronal cells. It is unclear whether this failure is due to the presence of a neuron-specific IkappaBalpha isoform or a defect in the MV signaling cascade that leads to IkappaBalpha phosphorylation and degradation. In this study, an IkappaBalpha-wild type (WT) expression vector was transfected into neuronal and glial cells and subsequently exposed to MV. In contrast to glial cells, IkappaBalpha-WT was degraded in neuronal cells in response to TNFalpha but not MV. The findings eliminate the existence of an IkappaBalpha isoform in neuronal cells that is resistant to phosphorylation by MV. Blocking de novo protein synthesis with cyclohexamide had no effect on neuronal IkappaBalpha, indicating that lack of degradation rather than increased synthesis is responsible for IkappaBalpha accumulation in MV-stimulated neuronal cells. To determine if malfunction in the MV receptor CD46 is responsible for failure of IkappaBalpha phosphorylation and degradation, neuronal cells were transfected with a wild type CD46 (CD46-WT) expression vector. MV stimulation of CD46-WT transfected cells failed to induce IkappaBalpha degradation. Collectively these findings indicate that failure of MV to phosphorylate neuronal IkappaBalpha is not due to a presence of an IkappaBalpha isoform or malfunction of the MV receptor, and is more likely to be due to a defect in the signaling pathway that normally leads to IkappaBalpha phosphorylation and degradation.

Primates exposed to cocaine in utero display reduced density and number of cerebral cortical neurons.

This study examined the effects of cocaine use during the second trimester of pregnancy on cerebral neocortical volume and density, and total number of neocortical neurons and glia in offspring. We also evaluated the extent of postnatal recovery of cytoarchitectural abnormalities previously observed in the neocortex of two-month-old primates born from cocaine-treated mothers (Lidow [1995] Synapse 21:332-334). Pregnant monkeys received cocaine orally (20 mg/kg/day) from the 40th to 102nd days of pregnancy (embryonic day [E]40-E102). On E64 and E65, the animals were injected with [(3)H]thymidine. Cerebral hemispheres of the offspring were examined at three years of age. We found a reduction in the neocortical volume and density and total number of neocortical neurons. The observed reduction in neuronal number within the neocortex was not accounted for by the increase in the number of neurons in the white matter of cocaine-exposed animals, because the number of these "extra" neurons was equal to only half that of missing neurons. We detected no significant changes in the number of neocortical glia. The cytoarchitectural abnormalities in the neocortex of prenatally cocaine-exposed three-year-old monkeys closely resembled previously described neocortical abnormalities in similarly exposed two-month-old animals: the neocortex lacked a discernible lamination; the majority of the cells labeled by [(3)H]thymidine injected during neocortical neurogenesis did not reach their proper position within the cortical plate. Therefore, postnatal maturation is not associated with significant improvement in neocortical organization in primates prenatally exposed to cocaine. There was, however, a postnatal recovery of low glial fibrillary acidic protein (GFAP) immunoreactivity previously observed in 2-month-old cocaine-exposed animals.

Effect of cocaine on cell proliferation in the cerebral wall of monkey fetuses.

This study examined the effect of cocaine on cell proliferation in the fetal monkey cerebral wall. Pregnant monkeys received cocaine daily (10 mg/kg, orally, in fruit treats, at 07.00 h and 19.00 h) beginning on the 40th day of pregnancy (E40). The control animals received fruit treats only. One set of monkeys was used to examine the state of cell proliferation in the fetal cerebral wall at peak cocaine levels. These animals were injected with [(3)H]thymidine intravenously on E73, 1.5 h after the morning drug or placebo administration. Another set of monkeys was used to determine the state of cell proliferation after cocaine concentration declined to ineffective levels. These animals were injected with [(3)H]thymidine on the same day of pregnancy 10 h after the treatment. Cesarean sections were performed 40 min after the radioisotope injection. The right hemispheres were processed for autoradiography. The left hemispheres were used for biochemical analysis of the radioisotope incorporation into DNA. The third set of monkeys was used to determine whether chronic cocaine treatment extends the timing of neocortical neuronogenesis. These monkeys received their final cocaine treatment on E102 (the last day of normal neocortical neuronogenesis) and were injected with [(3)H]thymidine 24 h later. On E113, the fetal brains were processed for emulsion autoradiography. We found a significant decrease in the density of [(3)H]thymidine-labeled cells and in the levels of this radioisotope incorporation into DNA in the fetal cerebral wall 1.5 h after cocaine administration. In contrast, 10 h after cocaine administration we detected a significantly elevated density of radiolabeled cells, and abnormally high levels of [(3)H]thymidine incorporation into DNA. This suggests that chronic intermittent administration of cocaine results in significant periodic fluctuations in cell production within the fetal cortical proliferative zones. We detected no cocaine-induced extension in neocortical neuronogenesis.

Cocaine affects cerebral neocortical cytoarchitecture in primates only if administered during neocortical neuronogenesis.

Previously, we demonstrated that chronic exposure of fetal monkeys to cocaine could result in development of the neocortex with significant cytoarchitectonic abnormalities [Synapse, 21 (1995) 435-444]. In the present study, we examined the developmental time-frame within which neocortical cytoarchitecture is susceptible to modifications by prenatal cocaine exposure. For this purpose, we assessed the integrity of cortical lamination and the position, density, and total number of occipital cortical neurons in 2-month-old monkeys which were prenatally exposed to chronic cocaine treatment either prior to the period of neocortical neuronogenesis, during the period of neocortical neuronogenesis, or after the period of neocortical neuronogenesis. We found that cocaine can interfere with the neocortical laminar organization and induce a reduction in the density and number of neocortical neurons only if it is administered at the time of neocortical neuronogenesis. During this window of vulnerability, an abnormal neocortex is generated as long as cocaine exposure is maintained, with corticogenesis becoming normal as soon as the administration of this drug is discontinued.

Pharmacokinetics of cocaine in maternal and fetal rhesus monkeys at mid-gestation.

We compared pharmacokinetics of cocaine and its metabolite, benzoylecgonine, in pregnant rhesus monkeys and their fetuses at mid-gestation: 1) after a single intravenous dose of cocaine, 2) after a single oral dose of cocaine, 3) after the last oral cocaine administration of a 50-day-long chronic cocaine treatment, and 4) on the last day of a 50-day-long chronic treatment with five daily intravenous cocaine injections. We found that intravenous administrations of cocaine produced maximal maternal levels of benzoylecgonine below the plasma levels for cocaine. In contrast, oral administrations resulted in the maximal maternal plasma levels of this metabolite significantly above those of cocaine. The bioavailability of the orally administered cocaine was calculated as 25%. Cocaine was detectable in the fetal plasma at maximal levels of approximately 1/5 of peak maternal levels for both single intravenous and single oral administrations. The maximal plasma levels of benzoylecgonine for the fetuses of the intravenously treated mothers were close to those of cocaine, whereas peak levels of this metabolite in the plasma of the fetuses of the mothers receiving the oral treatments were above those of cocaine. The chronic treatments resulted in significantly higher maximal levels of cocaine in the fetal circulation compared with those produced by single drug administrations.

Long-term effects of short-lasting early local inflammatory insult.

We examined the long-term effects of a short-lasting (approximately 24 h) inflammatory insult generated by injections of 0.25% carrageenan (1 microl/g) into the hindpaws of newborn (P0) rat pups. At P60 animals which experienced this early inflammatory insult showed significant alterations in the withdrawal responses to noxious stimulation of the affected paws. Furthermore, in the absence of ongoing inflammation, the withdrawal latencies to heat stimulation and withdrawal thresholds to mechanical stimulation were increased by such experience. In the presence of ongoing CFA-induced inflammation, however, the same early experience decreased these parameters of response to noxious stimulation. These data suggest that early inflammatory insult may differentially affect the aspects of nociceptive circuitry involved in transient pain sensitivity and in inflammation-induced hyperalgesia.

Antipsychotic treatment induces alterations in dendrite- and spine-associated proteins in dopamine-rich areas of the primate cerebral cortex.

BACKGROUND: Mounting evidence indicates that long-term treatment with antipsychotic medications can alter the morphology and connectivity of cellular processes in the cerebral cortex. The cytoskeleton plays an essential role in the maintenance of cellular morphology and is subject to regulation by intracellular pathways associated with neurotransmitter receptors targeted by antipsychotic drugs. METHODS: We have examined whether chronic treatment with the antipsychotic drug haloperidol interferes with phosphorylation state and tissue levels of a major dendritic cytoskeleton-stabilizing agent, microtubule-associated protein 2 (MAP2), as well as levels of the dendritic spine-associated protein spinophilin and the synaptic vesicle-associated protein synaptophysin in various regions of the cerebral cortex of rhesus monkeys. RESULTS: Among the cortical areas examined, the prefrontal, orbital, cingulate, motor, and entorhinal cortices displayed significant decreases in levels of spinophilin, and with the exception of the motor cortex, each of these regions also exhibited increases in the phosphorylation of MAP2. No changes were observed in either spinophilin levels or MAP2 phosphorylation in the primary visual cortex. Also, no statistically significant changes were found in tissue levels of MAP2 or synaptophysin in any of the cortical regions examined. CONCLUSIONS: Our findings demonstrate that long-term haloperidol exposure alters neuronal cytoskeleton- and spine-associated proteins, particularly in dopamine-rich regions of the primate cerebral cortex, many of which have been implicated in the psychopathology of schizophrenia. The ability of haloperidol to regulate cytoskeletal proteins should be considered in evaluating the mechanisms of both its palliative actions and its side effects.

Cocaine induces cell death within the primate fetal cerebral wall.

Transferase dUTP nick-end labelling (TUNEL) analysis was used to compare the occurrence of cell death in the cerebral wall of cocaine-exposed and drug-naïve monkey fetuses. The rhesus monkeys providing the drug-exposed fetuses received 10 mg/kg of cocaine orally (in fruit treats) in the morning and in the evening between pregnancy days 50 and 65. The control pregnant animals received fruit treats only. The fetuses were removed for analysis by Caesarean section 10 h after the last cocaine treatment. The sections of the cerebral wall from the cocaine-exposed fetuses contained significantly higher numbers of TUNEL-positive nuclei (counted either per section area or per 1000 unlabeled nuclei) than the matching sections from the drug-naïve fetuses. This elevation in the number of TUNEL-positive cells was observed through the entire depth of the fetal cerebral wall including its proliferative and intermediate zones, cortical plate and the marginal zone. The present study demonstrates that consumption of cocaine during pregnancy can result in increased occurrence of cell death in the developing cerebrum.

Projections of submucous neurons to the myenteric plexus in the guinea pig small intestine.

The distribution of submucous neurons that project to the myenteric plexus of the guinea pig small intestine was established by retrograde transport of the carbocyanine dye 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) from myenteric ganglia in organ culture in combination with immunohistochemistry. Following the application of DiI to the serosal surface of a single myenteric ganglion, from 2 to 15 DiI-labelled nerve cell bodies were labelled in the submucous plexus up to 7.9 mm circumferentially, 4.5 mm orally, and 3.4 mm aborally to the DiI application site. No cells were labelled in preparations in which connections between myenteric and submucous plexuses had been severed prior to DiI application. Cells that were immunoreactive for vasoactive intestinal polypeptide (VIP) or for substance P (SP) accounted for about 75% and 11% of DiI-labelled cells, respectively. Neither neuropeptide Y- nor calretinin-immunoreactive submucous neurons were labelled by DiI, indicating that these classes of neurons do not project to the myenteric plexus. Retrograde tracing from the myenteric plexus with Neurobiotin revealed that labelled VIP-immunoreactive neurons had several short, filamentous processes and a single long axon that could be followed through the circular muscle to myenteric ganglia without branches to the mucosa. The previously described projection of submucous, SP-immunoreactive putative sensory neurons to the myenteric plexus was confirmed. However, this study has identified a considerably larger population of presumed interneurons that are immunoreactive for VIP that likely transmit information from the submucous plexus to the myenteric plexus and presumably coordinate activity between the two ganglionated plexuses.

Characterization of myenteric interneurons with somatostatin immunoreactivity in the guinea-pig small intestine.

The projections, connections, morphology and electrophysiological features of the myenteric interneurons with somatostatin immunoreactivity in the guinea-pig small intestine have been established using retrograde tracing, immunohistochemistry, confocal microscopy and intracellular recording. After application of the fluorescent dye, 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI), to the myenteric plexus, up to 900 nerve cell bodies were labelled in each preparation. Somatostatin-immunoreactive neurons accounted for 13% of all retrogradely labelled cells and were located up to 70 mm orally. When DiI was applied to the submucous ganglia, many myenteric neurons were labelled and 8% of all retrogradely labelled cells were somatostatin immunoreactive and were located up to 60 mm oral to the DiI application sites. These neurons had ovoid cell bodies, a single axon, several long filamentous dendrites and received close contacts from 40-200 somatostatin-immunoreactive varicosities. Intracellular recordings revealed that these cells had features of both S (i.e. with Synaptic inputs) and AH (i.e. neurons with After Hyperpolarization) cells, receiving fast excitatory synaptic inputs, having characteristic "sag" in their response to hyperpolarizing current pulses and sometimes a long afterhyperpolarization following soma action potentials. It is concluded that somatostatin-immunoreactive neurons have distinct electrophysiological features and form very long anally directed interneuronal chains that connect with both myenteric and submucous neurons.

Immunohistochemical and electrophysiological characterization of submucous neurons from the guinea-pig small intestine in organ culture.

Immunohistochemical and electrophysiological properties of submucous neurons were investigated in organ cultures of the guinea-pig small intestine. Preparations of submucosa, with or without the myenteric plexus attached, were maintained in vitro for 3 to 5 days. Immunohistochemical labelling for peptides revealed that the cultured submucous plexus remained substantially intact and the immunoreactivity of cell bodies was well preserved. Substantial sprouting of nerve fibers immunoreactive for vasoactive intestinal peptide (VIP) or neuropeptide Y (NPY) was evident in submucous ganglia after 5 days in organ culture. Nerve fibers immunoreactive for substance P. somatostatin, 5-hydroxytryptamine or tyrosine hydroxylase were substantially depleted in submucous ganglia or perivascular nerves at 3 days and had virtually disappeared after 5 days in cultures of isolated submucosa. During intracellular recording from submucous neurons, action potentials were initiated by depolarizing current pulses in all neurons cultured with or without the myenteric plexus and muscle layers. Electrical stimulation of internodal strands evoked fast excitatory synaptic potentials (fast EPSPs) in nearly all neurons whether or not the myenteric plexus was present during the culture period up to 5 days. The removal of myenteric plexus and extrinsic nerves did not abolish fast EPSPs from submucous neurons, suggesting that some fast EPSPs may originate from neurons in the submucous plexus, although the possibility that new synapses formed by sprouting, or surviving axons severed from myenteric or sympathetic ganglia may have been functional, cannot be entirely excluded. This work demonstrates that the immunohistochemical and electrophysiological characteristics of submucous neurons are largely maintained in organ cultures of the submucosa.

Projections of specific morphological types of neurons within the myenteric plexus of the small intestine of the guinea-pig.

The projections of myenteric neurons within the myenteric plexus of the guinea-pig small intestine were established using retrograde tracing in organotypic culture. Three days after applying the fluorescent dye DiI to a single internodal strand in the myenteric plexus, 500-1000 nerve cell bodies were labelled. Of these, 77% were located oral to the application site, 15% were located anally and 7% were located within 1 mm of this site. Three major morphological types of neurons could be distinguished. Dogiel type I neurons had lamellar dendrites and single axons, Dogiel type II neurons had large smooth cell bodies and several long processes, and filamentous neurons had smooth ovoid cell bodies, single axons and several filamentous dendrites. Dogiel type I, II and filamentous neurons accounted for 54.6%, 38% and 7.4% of all filled cells, respectively. Labelled nerve cell bodies were present up to 13 mm aboral to the DiI application site; all neurons more than 2 mm aboral had Dogiel type I features. On the oral side, Dogiel type I neurons were found up to 110 mm, Dogiel type II neurons up to 100 mm and filamentous neurons up to 80 mm. Neurons with 2 mm oral or aboral to the DiI application site were located up to 7 mm circumferentially and were mainly Dogiel type II cells. This work revealed remarkable polarity within the myenteric plexus, with a significant prevalence of myenteric neurons projecting anally for longer distances than those projecting orally. These long pathways are probably involved in the coordination of intestinal motility.

Ultrastructural studies of the myenteric plexus and smooth muscle in organotypic cultures of the guinea-pig small intestine.

External muscle and myenteric plexus from the small intestine of adult guinea-pigs were maintained in vitro for 3 or 6 days. Myenteric neurons and smooth muscle cells from such organotypic cultures were examined at the electron-microscopic level. An intact basal lamina was found around the myenteric ganglia and internodal strands. Neuronal membranes, nuclei and subcellular organelles appeared to be well preserved in cultured tissues and ribosomes were abundant. Dogiel type-II neurons were distinguishable by their elongated electron-dense mitochondria, numerous lysosomes and high densities of ribosomes. Vesiculated nerve profiles contained combinations of differently shaped vesicles. Synaptic membrane specializations were found between vesiculated nerve profiles and nerve processes and cell bodies. The majority of nerve fibres were well preserved in the myenteric ganglia, in internodal strands and in bundles running between circular muscle cells. No detectable changes were found in the ultrastructure of the somata and processes of glial cells. Longitudinal and circular muscle cells from cultured tissue had clearly defined membranes with some close associations with neighbouring muscle cells. Caveolae occurred in rows that ran parallel to the long axis of the muscle cells. These results indicate that the ultrastructural features of enteric neurons and smooth muscle of the guinea-pig small intestine are well preserved in organotypic culture.

Long aboral projections of Dogiel type II, AH neurons within the myenteric plexus of the guinea pig small intestine.

Enteric AH neurons, with multipolar Dogiel type II morphology, project around the circumference of the intestine to myenteric ganglia, the submucosa and mucosa. Using retrograde labeling in vitro, intracellular recording, dye filling and immunohistochemistry, the projections of these neurons along the intestine were studied. When the retrograde tracer, Dil, was applied to the myenteric plexus, labeled nerve cell bodies were located up to 111 mm orally but only 13 mm aborally, demonstrating a marked difference in the lengths of projections up and down the small intestine. Of labeled nerve cell bodies located 2-110 mm orally, 43% had Dogiel type II morphology and of these, 70% were immunoreactive for calbindin, a calcium binding protein exclusive to Dogiel type II neurons. Intracellular filling with neurobiotin revealed several long circumferentially directed nerve fibers and short, filamentous dendrites; thus these were "dendritic" Dogiel type II neurons. This class accounts for approximately 3-4% of all myenteric neurons, and about 10% of all Dogiel type II neurons. Intracellular recordings revealed AH cell characteristics, with long afterhyperpolarizations following their action potentials, pronounced slow excitatory synaptic inputs and a lack of fast excitatory synaptic inputs. Antidromic action potentials could be evoked from the Dil application site in some cells, confirming their aboral projection. This is the first account of a major aboral projection of AH/Dogiel type II neurons and suggests an important role in aborally directed reflexes in the intestine.