In vivo Intracellular Recording of Neurons in the Supraoptic Nucleus of the Rat Hypothalamus

Intracellular recordings were made from cells in the hypothalamic supraoptic nucleus in the urethane-anaesthetized male rat using the ventral surgical approach. Impalements lasted from 5 min to 1 h and recorded cells had an input resistance of 55 to 170 megohms. Spikes of over 50 mV were recorded from 14 cells which could be antidromically activated by stimulation of the neural stalk. The spikes showed a hyperpolarizing afterpotential and the broadening characteristic of rapidly firing magnocellular neurons, which recovered rapidly (<200 ms). When depolarized, the cells showed evidence of a transient potassium current. Recurrent synaptic coupling between the recorded cell and adjacent cells would be expected to alter the hyperpolarizing afterpotential of an antidromic spike as compared with a spontaneous spike; no perceptible difference in the waveforms of the different types of spike could be detected in 11 spontaneously active cells. Application of just subthreshold stimuli to the neural stalk did not evoke depolarizing or hyperpolarizing potentials. Suprathreshold shocks to the neural stalk, when the antidromic spike was prevented by collision, also had no discernible effect on membrane potential. Thus intracellular recordings from magnocellular neurons in vivo revealed electrophysiological properties similar to those seen in vitro. No evidence for synaptic interconnection between magnocellular neurons was found in male rats.     

Triplet morbidity and mortality in a large case series.

OBJECTIVE: A significant increase in the triplet birth rate has occurred recently. This rise is of concern, as these infants are historically reported to be at risk of adverse outcome. Thus, we examined the outcome of triplet births in a large contemporary case series. STUDY DESIGN: Since 1993, detailed clinical data have been collected on all patients admitted to our Neonatal Intensive Care Unit. We retrospectively analyzed this database to examine triplet outcome. RESULTS: A total of 51 consecutive sets of triplets were born over a 9-year period. The mean birth weight for triplets was 1789+/-505 g, mean gestational age was 32.6+/-2.7 weeks, with discordancy present in 17.6% of neonates. Complications of prematurity were infrequent. Triplet survival to discharge was 96%. CONCLUSIONS: This large contemporary case series of triplets demonstrates excellent survival with low associated morbidity. These data suggest that there may no longer be medical justification for offering selective fetal reduction to parents with triplet pregnancies.     

Comparison of three intracellular markers for combined electrophysiological, morphological and immunohistochemical analyses.

Hypothalamic paraventricular and supraoptic neurons were recorded intracellularly in coronal slices and injected with Lucifer yellow, ethidium bromide or biocytin. Electrical properties, morphological staining and neurophysin immunohistochemistry were compared among the 3 markers. Lucifer yellow electrodes had a high resistance and frequently blocked during experiments. Neurons recorded with Lucifer yellow electrodes had low input resistances and low-amplitude, broad spikes. Lucifer yellow labeling in whole mount was highly fluorescent, revealing distal dendrites and axons. Of cells injected with Lucifer yellow, 64% were recovered but were faint after immunohistochemical processing. Recordings with ethidium bromide electrodes were similar to controls, although electrode blockage sometimes occurred. Only somata and proximal dendrites of ethidium bromide-filled neurons were visible in whole-mount. Forty percent of cells injected with ethidium bromide were recovered after immunohistochemical processing; these were invariably faint. Recordings with biocytin-filled electrodes were similar to control recordings. Biocytin-filled, HRP-labeled cells showed distal dendrites and often dendritic spines and axons in 50-75-microns sections. Seventy percent of biocytin-injected cells labeled with fluorescent markers were recovered and remained strongly labeled after immunohistochemical processing. Biocytin had the best electrical and staining properties for combined electrophysiological and anatomical studies.     

The recurrence of pain after neurosurgical procedures

Various reasons exist for the frequent failure of neurosurgical procedures to control pain. Three main classes of factors are involved in this failure—those relating to the surgeon and the surgery, those dependent on the disease, and those dependent on the nervous system itself. In the first class, technical and conceptual difficulties may explain the recurrence of pain after neurosurgery, including our incomplete understanding of the function of recognized pain pathways, the failure of the surgeon to apply known information, and technical limitations in our ability to carry out neurosurgery. Factors dependent upon the disease itself include alterations in the physical extent of the disease, and in the types of pain caused by it. Factors dependent on the nervous system include nerve and tract regeneration, changes in the anatomy of the CNS in response to both the disease and the neurosurgery, and, importantly, somatotopographic reorganization including the opening of alternative pathways.     

The regional vulnerability to hypoglycemia-induced neurotoxicity in organotypic hippocampal culture: protection by early tetrodotoxin or delayed MK-801.

Profound hypoglycemia selectively damages CA1 and the dentate gyrus of the hippocampus. We have examined the time course of hippocampal neuronal injury in organotypic cultures following in vitro "hypoglycemia," using the fluorescent vital dye propidium iodide to observe directly the regional distribution of early neuronal membrane injury in living cultures. The in vivo hippocampal pattern of hypoglycemic injury was reproduced by a 2 hr exposure to glucose-free media, which resulted in simultaneous, selective propidium staining of CA1 and the dentate gyrus starting by 4 hr after exposure. After 24 hr of recovery, CA3 remained spared. A similar pattern of propidium staining was produced by incubation of cultures for briefer periods in glucose-free medium containing 5 mM 2-deoxyglucose (2-DG) to inhibit glycolysis. This "hypoglycemic" pattern and time course of neuronal injury was mimicked by 300 microM aspartate but not by glutamate. The NMDA receptor antagonists MK-801 and CPP, but not the relatively selective non-NMDA receptor antagonist 6-cyano-7-dinitroquinoxaline-2,3-dione, prevented the development of propidium staining. MK-801 protected against injury even if added to the recovery media 30 min after the insult, while TTX (10 microM) protected only if added by the end of the exposure. The appearance of propidium staining after 4-6 hr of recovery was well correlated with histological observation of pyknotic neuronal nuclei in the injured regions. The characteristic hippocampal regional vulnerability of CA1 and the dentate gyrus to injury following profound hypoglycemia can be reproduced in organotypic hippocampal culture and appears to be mediated both by an early TTX-sensitive component and by a more prolonged period of toxic NMDA receptor activation, extending for at least 30 min into the recovery period.     

Identification and characterization of neurons with tremor-frequency activity in human globus pallidus

 Many previous studies have demonstrated the existence of neurons with tremor-frequency activity (”tremor cells”) in the thalamus of Parkinson’s disease (PD) patients and these neurons are presumed to play a role in the pathogenesis of tremor. Since a major input to motor thalamus (Voa and Vop) is from the internal segment of the globus pallidus (GPi), neurons with tremor-frequency activity in motor thalamus may receive input from neurons in GPi. The aim of this study was to quantify the characteristics of tremor cells in human globus pallidus. In three PD patients with tremor undergoing microelectrode exploration of the globus pallidus prior to pallidotomy, 228 neurons were sampled, and 28 (12.3%) were identified to fire at the same frequency as the tremor. These ”tremor cells” were located in the ventral portion of GPi. Autocorrelogram analysis of the sampled spike trains of these 28 tremor cells was carried out over sequential 10-s time segments, and autocorrelograms showing maximal oscillatory activity were graded from 0 to 10. Average tremor cell oscillation grades ranged from 6.8 to 7.8, similar to those reported in the MPTP-induced primate model of parkinsonism. The average tremor cell oscillation grade varied between patients, as did the clinical measures of tremor severity. Tremor cells had oscillations in spike discharges at the same average frequency (4.2–5.2 Hz) as the patient’s tremor determined from the electromyogram and accelerometry records of one or more limbs (4.0–5.4 Hz), and the individual values were correlated (r ²=0.73) over the total range (3.7–5.6 Hz). The results of this study demonstrate the presence of neurons with 4–6 Hz tremor-frequency activity in GPi, supporting a role of the globus pallidus in the production of rest tremor in PD patients. Received: 27 February 1996 / Accepted: 18 October 1996     

Antigen receptor signalling in apoptosis-resistant mutants of WEHI 231 cells

Ligation of membrane immunoglobulin M (mIgM) induces cell cycle arrest and apoptosis in the WEHI 231 B-lymphoma cell line. The molecular mechanisms which link receptor ligation and the nuclear events that underlie this response, have yet to be fully elucidated. Here we have examined the signals induced following mIgM cross-linking in variants of WEHI 231 that no longer undergo apoptosis in response to this stimulus. Tyrosine phosphorylation of cellular substrates in two of the variants is identical to that seen in wild-type cells but in one of the mutants, VS2.12, a restricted set of substrates becomes tyrosine phosphorylated. In a second variant (E8), mIgM cross-linking does not induce elevation of intracellular Ca2+, although tyrosine phosphorylation of PLCgamma2 is induced to an equivalent extent to that seen in WEHI 231 cells. A third variant, 2E10.F9, is resistant to apoptosis despite the fact that all signals analysed appear to be similar to those induced in wild-type cells. Our findings show that resistance to apoptosis can arise as a result of mutations affecting discrete stages of the mIgM signalling pathway. The mutant lines reported here show defects that have not yet been identified in previous studies and are likely to be useful tools in dissecting the signalling of cell death in B lymphocytes.