gamma-Aminobutyric acid (GABA), the principal inhibitory neurotransmitter, activates a persistent low amplitude tonic current in several brain regions in addition to conventional synaptic currents. Here we demonstrate that GABA(A) receptors mediating the tonic current in hippocampal neurons exhibit functional and pharmacological properties different from those of quantal synaptic currents. Patch-clamp techniques were used to characterize miniature inhibitory postsynaptic currents (mIPSCs) and the tonic GABAergic current recorded in CA1 pyramidal neurons in rat hippocampal slices and in dissociated neurons grown in culture. The competitive GABA(A) receptor antagonists, bicuculline and picrotoxin, blocked both the mIPSCs and the tonic current. In contrast, mIPSCs but not the tonic current were inhibited by gabazine (SR-95531). Coapplication experiments and computer simulations revealed that gabazine bound to the receptors responsible for the tonic current but did not prevent channel activation. However, gabazine competitively inhibited bicuculline blockade. The unitary conductance of the GABA(A) receptors underlying the tonic current (approximately 6 pS) was less than the main conductance of channels activated during quantal synaptic transmission (approximately 15--30 pS). Furthermore, compounds that potentiate GABA(A) receptor function including the benzodiazepine, midazolam, and anesthetic, propofol, prolonged the duration of mIPSCs and increased tonic current amplitude in cultured neurons to different extents. Clinically-relevant concentrations of midazolam and propofol caused a greater increase in tonic current compared with mIPSCs, as measured by total charge transfer. In summary, the receptors underlying the tonic current are functionally and pharmacologically distinct from quantally activated synaptic receptors and these receptors represent a novel target for neurodepressive drugs.

Healthy bullfrog sympathetic ganglion cells often show a two-component afterhyperpolarization (AHP). Both components can be reduced or abolished by adding Cachannel blockers or by removing external Ca. Application of a single electrode "hybrid clamp"-i.e., switching from current-to voltage-clamp at the peak of the AHP, reveals that the slow AHP component is generated by a small, slow, monotonically decaying outward current, which we call Ihm. IAHP is blocked by Ca-removal or by apamin and is a pure K current. It is slightly sensitive to muscarine and to tetraethylammonium ion but is much less so than muscarine-sensitive (IM) and fast Ca-dependent (Ic) K currents. It also can be recorded in dualelectrode voltage-clamp experiments, where it is seen as a slow, small component of the outward tail current that follows brief depolarizations to 0 mV or beyond. Ic is seen as an early, fast, large component of the same tail current. Both components are blocked by Ca removal, but only the Ic component is blocked by low doses of tetraethylammonium ion. Thus, bullfrog ganglion cells exhibit two quite distinct Ca-dependent K currents, which differ in size, voltage-sensitivity, kinetics, and pharmacology. These two currents also play quite separate roles in shaping the action potential.Recent voltage-clamp work in bullfrog sympathetic ganglion cells (1-3) has established the existence of a large, rapid Caactivated K current that is partly responsible for spike repolarization and early afterhyperpolarization (AHP). This current, which we term Ic, has several hallmarks: sensitivity to tetraethylammonium ion (Et4N+); insensitivity to muscarine, apamin, and barium; and a strong voltage dependence.It is carried by high-conductance channels like those in chromaffin cells and myotubes (4, 5). However, carefully impaled bullfrog neurons often show a second slow component of the afterhyperpolarization, which, while dependent on Ca-influx, seems to be inhibited by muscarinic-receptor activation (6) and rather insensitive to membrane potential (7). We have investigated this slow AHP component using voltage-clamp techniques, and we find that it is due to a Cadependent K current that, while it has some similarities to both Ic and the muscarine-sensitive K current, which we call IM (8)

In pancreatic ␤-cells, glucose metabolism signals insulin secretion by altering the cellular array of messenger molecules. ATP is particularly important, given its role in regulating cation channel activity, exocytosis, and events dependent upon its hydrolysis. Uncoupling protein (UCP)-2 is proposed to catalyze a mitochondrial inner-membrane H ؉ leak that bypasses ATP synthase, thereby reducing cellular ATP content. Previously, we showed that overexpression of UCP-2 suppressed glucose-stimulated insulin secretion (GSIS) in isolated islets (1). The aim of this study was to identify downstream consequences of UCP-2 overexpression and to determine whether insufficient insulin secretion in a diabetic model was correlated with increased endogenous UCP-2 expression. In isolated islets from normal rats, the degree to which GSIS was suppressed was inversely correlated with the amount of UCP-2 expression induced. Depolarizing the islets with KCl or inhibiting ATP-dependent K ؉ (K ATP ) channels with glybenclamide elicited similar insulin secretion in control and UCP-2-overexpressing islets. The glucose-stimulated mitochondrial membrane (⌿ m ) hyperpolarization was reduced in ␤-cells overexpressing UCP-2. ATP content of UCP-2-induced islets was reduced by 50%, and there was no change in the efflux of Rb ؉ at high versus low glucose concentrations, suggesting that low ATP led to reduced glucose-induced depolarization, thereby causing reduced insulin secretion. Sprague-Dawley rats fed a diet with 40% fat for 3 weeks were glucose intolerant, and in vitro insulin secretion at high glucose was only increased 8.5-fold over basal, compared with 28-fold in control rats. Islet UCP-2 mRNA expression was increased twofold. These studies provide further strong evidence that UCP-2 is an important negative regulator of ␤-cell insulin secretion and demonstrate that reduced ⌬⌿ m and increased activity of K ATP channels are mechanisms by which UCP-2-mediated effects are mediated. These studies also raise the possibility that a pathological upregulation of UCP-2 expression in the prediabetic state could contribute to the loss of glucose responsiveness observed in obesity-related type 2 diabetes in humans.

Pain education, especially for undergraduates, has been identified as important to changing problematic pain practices, yet, no published data were found describing an integrated, interprofessional pain curriculum for undergraduate students. Therefore, this project aimed to develop, implement, and evaluate an integrated pain curriculum, based on the International Association for the Study of Pain curricula [http://www.iasp-pain.org/curropen.html], for 540 students from six Health Science Faculties/Departments. Over an 18-month period, the University of Toronto Centre for the Study of Pain's Interfaculty Pain Education Committee developed a 20-h undergraduate pain curriculum to be delivered during a 1-week period. Students from Dentistry, Medicine, Nursing, Pharmacy, Physical Therapy, and Occupational Therapy participated as part of their 2nd or 3rd year program. Teaching strategies included large and small groups, Standardized Patients, and 63 facilitators. Evaluation methods included: (a) pre- and post-tests of the Pain Knowledge and Beliefs Questionnaire (PKBQ) and (b) Daily Content and Process Questionnaire (DCPQ) to obtain feedback about process, content, and format across the curriculum's 5 days. A significant improvement in pain knowledge and beliefs was demonstrated (t = 181.28, P < 0.001), although non-responders were problematic at the post-test. DCPQ overall ratings of 'exceeding or meeting expectations' ranged from 74 to 92%. Ratings were highest for the patient-related content and panel, and the small-group discussions with Standardized Patients. Overall evaluations were positive, and statistically significant changes were demonstrated in students' pain knowledge and beliefs. This unique and valuable learning opportunity will be repeated with some modifications next year.

Propofol (2,6 di-isopropylphenol) is an alkyphenol recently introduced for use as a general anesthetic. The modulation of GABAA receptor activation and desensitization by propofol was studied using a rapid perfusion system and whole-cell voltage-clamp recordings from mouse hippocampal neurons. The effects of concentrations of propofol used clinically on single-channel and synaptic currents were also examined. Propofol evoked current responses (EC50 = 61 microM) and shifted the dose-response curve of GABA-activated current to the left without altering the maximum of the GABA response. Preincubation with propofol and GABA led to desensitization of the GABA response (EC50 = 454 microM and 23 microM, respectively). Saturating concentrations of GABA (600 microM) evoked currents that peaked and then declined in a biexponential fashion with fast and slow time constants of tau f = 1.0 sec and tau s = 3.5 sec. Propofol (10 microM) did not change the amplitude of the peak response but decreased the rates of decay approximately 1.5-fold and enhanced the steady-state current proportionately. Recovery from desensitization was also biexponential (tau f = 11 sec, tau s = 69 sec) but not influenced by propofol. Single-channel recordings from outside-out patches demonstrated that both propofol and GABA activated channels with a 30 pS and 21 pS open state. Propofol increased the frequency but not the duration or conductance of GABA-activated events. Miniature inhibitory postsynaptic currents (mlPSCs) were evoked by the application of hypertonic sucrose to the cell soma. Propofol (2 microM) prolonged the decay time of mlPSCs to an extent similar to which it increased the open probability of GABA-activated channels (2.3- vs 3-fold). A sequential model, based on a previous scheme of GABA receptor gating (Weiss and Magelby, 1989), is presented to summarize propofol's actions on GABAA receptor function. We show through simulation that the model reliably reproduced the whole-cell tracings. Our results indicate that propofol's neurodepressive actions will be associated with enhancement of inhibitory synaptic transmission.

Propofol (2,6-di-isopropylphenol) has multiple actions on GABA(A) receptor function that act in concert to potentiate GABA-evoked currents. To understand how propofol influences inhibitory IPSCs, we examined the effects of propofol on responses to brief applications of saturating concentrations of GABA (1-30 mM). GABA was applied using a fast perfusion system to nucleated patches excised from hippocampal neurons. In this preparation, propofol (10 microM) had no detectable agonist effect but slowed the decay, increased the charge transfer (62%), and enhanced the peak amplitude (8%) of currents induced by brief pulses (3 msec) of GABA. Longer pulses (500 msec) of GABA induced responses that desensitized with fast (tau(f) = 1.5-4.5 msec) and slow (tau(s) = 1-3 sec) components and, after the removal of GABA, deactivated exponentially (tau(d) = 151 msec). Propofol prolonged this deactivation (tau(d) = 255 msec) and reduced the development of both fast and slow desensitization. Recovery from fast desensitization, assessed using pairs of brief pulses of GABA, paralleled the time course of deactivation, indicating that fast desensitization traps GABA on the receptor. With repetitive applications of pulses of GABA (0.33 Hz), the charge transfer per pulse declined exponentially (tau approximately 15 sec) to a steady-state value equal to approximately 40% of the initial response. Despite the increased charge transfer per pulse with propofol, the time course of the decline was unchanged. These experimental data were interpreted using computer simulations and a kinetic model that assumed fast and slow desensitization, as well as channel opening developed in parallel from a pre-open state. Our results suggest that propofol stabilizes the doubly liganded pre-open state without affecting the isomerization rate constants to and from the open state. Also, the rate constants for agonist dissociation and entry into the fast and slow desensitization states were reduced by propofol. The recovery rate constant from fast desensitization was slowed, whereas that from slow desensitization appeared to be unchanged. Taken together, the effects of propofol on GABA(A) receptors enhance channel opening, particularly under conditions that promote desensitization.

1. Mouse hippocampal neurons grown in dissociated cell culture were patch clamped using a whole cell voltage clamp (discontinuous switching clamp) technique. The currents generated by pressure applications of excitatory amino acids were studied over a wide range of holding potentials, and current-voltage curves were plotted. Excitatory amino acids that activated the N-methyl-D-aspartic acid (NMDA) receptor demonstrated some degree of desensitization with repeated applications, whereas the currents observed in response to kainic acid (KAI) did not. Desensitization could be minimized by keeping the frequency of application sufficiently low (i.e., less than 0.1 Hz). 2. The short-acting dissociative anaesthetic, ketamine (2-50 microM), selectively blocked L-aspartic acid (L-Asp), NMDA, and L-glutamic acid (L-Glu) currents while sparing those in response to KAI. Therefore, ketamine is a relatively selective blocker of the NMDA response versus that (those) activated by KAI. 3. The block by ketamine of excitatory amino acid currents is highly voltage dependent. Concentrations of ketamine that had little effect on outward current responses at depolarized potentials were quite effective at blocking inward current responses at hyperpolarized potentials. In contrast, DL-2-amino-5-phosphonovaleric acid (APV) was equally effective at blocking both inward and outward currents (voltage independent). The voltage dependence of ketamine (a positively charged molecule) could be accounted for if ketamine blocked the NMDA response by binding to a site that experienced 55% of the membrane field. 4. In the presence of ketamine, peak inward currents evoked by repeated applications of NMDA, L-Asp, or L-Glu progressively declined to a steady-state level of block (use-dependent block). This decrement occurred at frequencies much lower than those that were employed to demonstrate desensitization (in the absence of ketamine). Moving the membrane potential to depolarized values did not, in itself, relieve the ketamine block. However, if the appropriate excitatory amino acid (L-Asp, NMDA, L-Glu) was applied during the period of depolarization, a relief of the block could be demonstrated. No recovery from the blockade occurred with periods of rest (no amino acid application) as long as 5 min. Furthermore, no recovery was observed even when ketamine was washed out of the bathing solution until the appropriate agonist was applied. Thus recovery from blockade, like development of blockade, was use dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

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