Development of action potentials and apamin-sensitive after-potentials in mouse vestibular nucleus neurones

The postnatal maturation of medial vestibular nucleus (MVN) neurones was examined in slices of the dorsal brainstem prepared from balb/c mice at specific stages during the first postnatal month. Using spike-shape averaging to analyse the intracellularly recorded action potentials and after-hyperpolarisations (AHPs) in each cell, all the MVN neurones recorded in the young adult (postnatal day 30; P30) mouse were shown to have either a single deep AHP (type A cells), or an early fast and a delayed slow AHP (type B cells). The relative proportions of the two subtypes were similar to those in the young adult rat. At P5, all the MVN cells recorded showed immature forms of either the type A or the type B action potential shape. Immature type A cells had broad spontaneous spikes, and the characteristic single AHP was small in amplitude. Immature type B cells had somewhat narrower spontaneous spikes that were followed by a delayed, apamin-sensitive AHP. The delayed AHP was separated from the repolarisation phase of the spike by a period of isopotentiality. Over the period P10–P15, the mean resting potentials of the MVN cells became more negative, their action potential fall-times became shorter, the single AHP in type A cells became deeper, and the early fast AHP appeared in type B cells. Until P15 cells of varying degrees of electrophysiological maturity were found in the MVN but by P30 all MVN cells recorded were typical adult type A or type B cells. Exposure to the selective blocker of SK-type Ca-activated K channels, apamin (0.3 μM), induced depolarising plateaux and burst firing in immature type B cells at rest. The duration of the apamin-induced bursts and the spike frequency during the bursts were reduced but not abolished after blockade of Ca channels in Ca-free artificial cerebrospinal fluid containing Cd²⁺. By contrast, in mature type B cells at rest apamin selectively abolished the delayed slow AHP but did not induce bursting activity. Apamin had no effect on the action potential shape of immature type A cells. These data show that the apamin-sensitive I _AHP is one of the first ionic conductances to appear in type B cells, and that it plays an important role in regulating the intrinsic rhythmicity and excitability of these cells. Received: 19 November 1996 / Accepted: 30 June 1997     

Effect of glycine and glycine receptor antagonists on NMDA-induced brain injury

In postnatal day 7 rats, a unilateral intrastriatal injection of 12.5 nmol of N-methyl-D-aspartate (NMDA) reproducibly injures the ipsilateral striatum, adjacent hippocampus and overlying cortex. The severity of injury can be quantified by comparing cerebral hemisphere weights in animals sacrificed 5 days after the injection. Co-injection of NMDA and the glycine receptor antagonists kynurenic acid (KYN) or 7-chlorokynurenic acid (7-CKA) reduced the severity of NMDA-induced damage in a dose-dependent fashion. One hundred nmol of KYN with 12.5 nmol of NMDA reduced average % damage from 19.3 +/- 0.9% (n = 9) to 2.3 +/- 0.5% (n = 6), P less than 0.001, ANOVA. Co-injection of 40 nmol of 7-CKA with 12.5 nmol of NMDA (n = 6) reduced average % damage from 17.1 +/- 1.6% (n = 15) to 3.0 +/- 0.6%, P less than 0.001, ANOVA. Concurrent injection of 1000 nmol glycine with 5 nmol NMDA did not increase the extent of NMDA-induced damage. Our results demonstrate that glycine receptor antagonists attenuate NMDA-induced brain injury in vivo.     

Monocyte chemotactic protein-3 (MCP3) interacts with multiple leukocyte receptors: binding and signaling of MCP3 through shared as well as unique receptors on monocytes and neutrophils

The diversity of monocyte chemotactic protein (MCP)3 target cell types, as well as the capacity of MCP3 to desensitize leukocyte responses to other CC chemokines, suggested that MCP3 may interact with multiple CC chemokine receptors. The purpose of this study is to establish how MCP3 binds and activates monocytes and neutrophils. We show that human monocytes exhibit high-affinity binding for ¹²⁵I-MCP3 with an estimated Kd of 1–3 nM and about 10000 binding sites/cell. The binding of ¹²⁵I-MCP3 to monocytes was progressively less well competed by CC chemokines macrophage inflammatory protein (MIP)lα (Kd = 5–10 nM), RANTES (Kd = 5–10 nM), MCP1 (monocyte chemoattractant and activating factor, or MCAF) (Kd = 60 nM) and MIP1β (Kd > 100 nM). On the other hand, unlabeled MCP3 displaced the binding of radiolabeled MIP1α, RANTES, MCP1 and MIP1β as effectively as the isologous CC chemokines. In agreement with the binding data, pretreatment of monocytes with MCP3 completely desensitized the calcium flux in response to MIP1α and RANTES. However, MIP1α and RANTES failed to desensitize the response of monocytes to MCP3. MCP3 and MCP1 partially desensitized each other's effects on monocytes. These binding and cross-desensitization results suggest that MCP3 binds and signals through other binding sites in addition to those shared with MIP1α, RANTES and MCP1. The unidirectional competition for MIP1β binding and signaling by MCP3 suggests the existence of an as-yet unidentified site for MCP3 shared with MIP1β. The existence of another unique binding site(s) for MCP3 was further shown by the failure of any of the other CC chemokines to compete effectively for MCP3 binding on neutrophils. MCP3 in our study was also the only human CC chemokine that consistently chemoattracted neutrophils. These results suggest that MCP3 is a ligand that can bind and activate a broad range of target cells through receptors shared by other CC chemokines as well as its own receptor.     

Rapid intraoperative immunohistochemical evaluation of sentinel lymph nodes for metastatic breast carcinoma.

OBJECTIVE: Sentinel lymph node (SLN) biopsy is an integral part of the surgical management of patients with breast cancer. Rapid immunohistochemistry (RIHC) has the potential to increase detection of metastatic carcinoma at the time of frozen section consultation. The authors assessed the accuracy and turnaround time of a newly developed RIHC method for pancytokeratin (RIHC-CK). METHODS: Sixty-six SLNs from 32 patients with breast carcinoma were examined for metastasis using the Zymed Sentinel Lymph Node Rapid IHC Kit. Intraoperative frozen sections (6 mum) of the SLNs were incubated with Zymed anti-pan-cytokeratin/HRP conjugate, diaminobenzidine (DAB), and stained with hematoxylin. Slides were ready within 8 minutes and were interpreted as positive or negative for metastatic carcinoma. Results were compared with previous intraoperative touch preparations, frozen sections, hematoxylin and eosin (Perm H&E), and AEl/3-immunostained permanent sections (Perm CK). RESULTS: Fourteen lymph nodes (19%) in 13 patients tested positive for metastatic carcinoma in Perm H&E, the gold standard. RIHC-CK had the highest sensitivity (92%) of the intraoperative tests, compared with touch preparations (64%) and frozen sections (80%). RIHC-CK showed 94% accuracy, compared with 96% (frozen section) and 93% (touch preparation). The RIHC technique took 8 minutes and was easy to perform and interpret. CONCLUSIONS: Zymed RIHC is a sensitive method for detecting breast cancer metastases in SLNs. The speed, accuracy, and ease of interpretation of the test allow for recognition of micrometastases (<2 mm) that might otherwise be undetectable by current methods of intraoperative evaluation. The prognostic significance and effect on surgical management of micrometastases in SLNs have yet to be determined.     

Tizanidine (DS103-282), a centrally acting muscle relaxant, selectively depresses excitation of feline dorsal horn neurones to noxious peripheral stimuli by an action at alpha 2-adrenoceptors

The effects of microiontophoretic ejection of tizanidine were compared with those of adrenoceptor agonists on responses of single laminae IV and V neurones to noxious and innocuous cutaneous stimuli. Tizanidine, noradrenaline and clonidine depressed neuronal responses to noxious but not innocuous stimuli. Spontaneous activity was also depressed by these three substances. By contrast, beta- and alpha 1-adrenoceptor agonists had no consistent effect on neuronal responses to cutaneous stimuli. The selective actions of tizanidine, noradrenaline and clonidine were reversibly antagonized by the alpha 2-adrenoceptor antagonist RX781094 but not by WB4101 (alpha 1 antagonist). The binding of an alpha 2-adrenoceptor ligand to rat brain membranes was preferentially displaced by tizanidine. These results indicate an interaction of tizanidine with central alpha 2-adrenoceptors.     

Angiokeratoma corporis diffusum with features of a mucopolysaccharidosis.

Two cases of angiokeratoma corporis diffusum with mental retardation and some features of a mucopolysaccharidosis have been investigated biochemically, histopathologically, and by electron microscopy. It is submitted, on this evidence, that they are examples of a hitherto undescribed form of lysosomal enzyme deficiency disease.     

Effects of potassium channel blockers on baclofen-induced suppression of paroxysmal discharges in rat neo-cortical slices

Baclofen reduced the frequency and aborted the bursts of spontaneous paroxysmal discharges in rat neocortical slices maintained in magnesium-free medium. This action was prevented by pretreatment with barium or caesium, which each increased the ictaform burst frequency, amplitude and duration. 4-Amino-pyridine also increased the burst frequency but reduced the amplitude and did not completely prevent the action of baclofen. Evidently baclofen suppresses such discharges by opening potassium channels normally involved in limiting the burst activity.