William R. Proctor

The role of cyclic AMP as a precursor of extracellular adenosine in the rat hippocampus

Extracellular adenosine 3:5-cyclic monophosphate (cAMP) is a potential source of the inhibitory neuromodulator adenosine in the brain. Previous work has demonstrated that cAMP, which is formed intracellularly, can be transported into the extracellular space and subsequently catabolized to adenosine. However, the physiological conditions under which cAMP release might lead to adenosine formation and activation of adenosine receptors are not well understood. In this study we demonstrate that superfusion of hippocampal slices with cAMP or forskolin led to the formation of extracellular adenosine which activated adenosine receptors in a manner comparable to that seen with adenosine superfusion. In contrast, application of brief pulses of cAMP onto the cell bodies of CA1 pyramidal neurons failed to produce an adenosine receptor-mediated response, while application of brief pulses of adenosine or AMP elicited significant responses. These data suggest that large, prolonged increases in extracellular cAMP levels can result in the formation of extracellular adenosine and the activation of adenosine receptors, but brief increases in cAMP levels in the vicinity of individual neurons cannot. These findings imply that increases in cAMP levels may lead to relatively slow increases in extracellular adenosine, as opposed to the fast, spatially restricted increases that would occur following the release of other adenine nucleotides.

Ethanol enhances synaptically evoked GABAA receptor-mediated responses in cerebral cortical neurons in rat brain slices

Previous intracellular electrophysiological studies on rat hippocampal brain slices have shown very little effect of acute ethanol application on synaptically evoked GABAA receptor-mediated responses recorded in CA1 pyramidal neurons. The present study was designed to compare the effects of ethanol on pyramidal neurons in the hippocampus and cerebral cortex. Using conventional intracellular microelectrodes (60-80 M omega) to impale cortical neurons in brain slices, 80 mM ethanol application did not affect the membrane input impedance nor evoked EPSPs, but significantly affected the resting membrane potential (usually a 2-5 mV hyperpolarization). When stimulus-evoked GABAA-mediated IPSCs were studied using whole-cell recordings from cortical neurons voltage-clamped at depolarizing potentials, monophasic IPSCs were evoked that were blocked by bicuculline, increased by pentobarbital, and enhanced by ethanol superfusion in a dose dependent manner over the range of 20-160 mM. Hippocampal IPSCs recorded under identical conditions were not enhanced by ethanol. Parallel studies of GABA-stimulated 36Cl- flux measurements in microsacs prepared from hippocampal, cerebral cortical and cerebellar tissue demonstrated that ethanol significantly enhanced (30-50%) 36Cl- flux in microsacs derived from the cerebral cortex and cerebellum, but not in microsacs prepared from the hippocampus. These results demonstrate that there are clear brain region-dependent differences in the way that GABAA receptor function is altered by acute ethanol, and that these differences are apparent not only as an enhancement of responses to exogenous GABA, but also as a facilitation of the responses to endogenous GABA released from inhibitory nerve terminals during synaptic activation.

Dissociation of μ and δ opioid receptor-mediated reductions in evoked and spontaneous synaptic inhibition in the rat hippocampus in vitro

Modulation of gamma-aminobutyric acid (GABA)-mediated inhibition, and glutamate-mediated excitation by highly selective mu and delta opioid agonists was studied using intracellular recordings of CA1 pyramidal neuron synaptic responses in superfused hippocampal slices. Equimolar concentrations of the selective mu agonist, [Tyr-(D-Ala)-Gly-(N-Me-Phe)-Gly-ol]-enkephalin (DAGO), or the delta selective agonist, [D-Pen2,D-Pen5]-enkephalin (DPDPE), reversibly increased the amplitudes of excitatory post-synaptic potentials (EPSPs), evoked by Schaffer collateral/commissural stimulation, without altering the input resistance or resting membrane potential of these CA1 pyramidal neurons. The increased EPSP amplitudes resulting from superfusion with the enkephalin analogs were qualitatively similar to those caused by the GABAA receptor antagonist, bicuculline methiodide (BMI). Specific stimulation/recording protocols and micro-lesions of the slices were used to evoke relatively pure forms of recurrent and feed-forward GABA-mediated inhibitory post-synaptic potentials (IPSPs). The mu opioid agonist DAGO reduced both recurrent and feed-forward IPSPs, while the delta agonist DPDPE had no effect upon these responses. To test the hypothesis that the enhancement of pyramidal neuron EPSPs by delta (and mu) opioids was due to the reduction of an inhibitory potential that was coincident with the EPSP, DPDPE or the mu agonist, DAGO, were applied while recording monosynaptic IPSPs following the elimination of EPSPs by the glutamate receptor antagonists, D,L-2-amino-5-phosphonovalerate (APV) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). The mu agonist, DAGO, reversibly reduced these pharmacologically isolated IPSPs, while the delta agonist, DPDPE, had no effect upon these responses. Despite the fact that the delta agonist, DPDPE, had no effect on recurrent, feed-forward or monosynaptic evoked IPSPs, this enkephalin did reversibly reduce the frequency of spontaneously occurring IPSPs, measured using whole-cell recordings with pipettes containing 65 mM KCl. The mu agonist, DAGO, and the GABAA antagonist, BMI, similarly reduced spontaneous IPSP rates. We conclude from these data that mu and delta opioid receptor activation increases EPSPs via the reduction of a form of GABAergic inhibition that is difficult to characterize, and which may be distinct from conventional feed-forward and recurrent inhibition. Furthermore, delta opioids seem to reduce this form of GABAergic inhibition selectively, while mu opioids reduced this inhibition, and conventional feed-forward and recurrent IPSPs as well.

Local anaesthetic actions of cocaine: effects on excitatory and inhibitory synaptic responses in the hippocampus in vitro

1 The basis for the proconvulsant action of cocaine was investigated in the CA1 region of the rat hippocampal slice in vitro. 2 Superfusion with 100 μm cocaine depressed inhibitory and excitatory postsynaptic potentials recorded intracellularly from CA1 pyramidal neurones; both types of potentials were inhibited to an equal extent. When inhibition was assessed using extracellular recording of population spike responses before and after conditioning impulses, there did not appear to be any selective effect upon either recurrent or feed‐forward γ‐aminobutyric acid (GABA)ergic inhibition. 3 Not all responses showed equivalent sensitivity to the local anaesthetic actions of cocaine. In particular, the antidromic population spike evoked by stimulation of the alveus was significantly more sensitive than the presynaptic fibre spike elicited by stimulation of stratum radiatum. 4 The rate of interictal spiking in hippocampus, induced by penicillin and increased potassium in the perfusion medium, was depressed by superfusion with cocaine in the range 5–100 μm. 5 These results suggest that cocaine does not have a selective depressant effect upon inhibitory pathways in the CA1 region of the hippocampus. Although the hippocampus shows epileptiform activity following systemic administration of local anaesthetics such as cocaine in the intact rat, this effect may not reflect a direct hippocampal site of drug action.

Bremazocine differentially antagonizes responses to selective μ and δ opioid receptor agonists in rat hippocampus

1 The effects of μ, δ and κ opioid receptor agonists were examined on evoked field potentials in brain slices prepared from rat hippocampus. 2 The effects of the μ‐selective opioid peptide [d‐Ala2, NMe‐Phe4, Met(O)5ol]enkephalin (FK 33–824) and the δ‐selective peptide [d‐Pen2, d‐Pen5]enkephalin (DPDPE) were qualitatively and quantitatively similar. Both increased the amplitude of evoked population spike responses when perfused in low nanomolar concentrations in a fashion consistent with what has been previously reported for other opiate agonists such as morphine. The κ‐selective agonists bremazocine and U‐50, 488H were without effect upon evoked responses at concentrations as high as 10 μm. 3 Bremazocine, but not U‐50, 488H, proved to be an extremely potent antagonist of responses to both μ‐and δ‐selective agonists. Moreover, bremazocine was considerably more potent in antagonizing responses to FK 33–824 than DPDPE, which supports the hypothesis that FK 33–824 and DPDPE act via different receptors. Thus, although bremazocine is an agonist at κ receptors, it appears to act as an antagonist at other opioid receptor sites.

Ethanol selectively enhances the hyperpolarizing component of neocortical neuronal responses to locally applied GABA

Local application of GABA to rat cerebral cortical neurons in brain slices elicited biphasic responses mediated via GABAA receptors. The fast component of the response, which was most apparent with somatic application of GABA, was hyperpolarizing at the normal resting membrane potential (GABAh response). The slower component could be elicited by GABA application to nearly all regions of the cell, and was depolarizing at the resting membrane potential (GABAd response). The reversal potential of evoked IPSCs recorded with whole-cell patch electrodes (-68 mV) was comparable to the reversal potential of the GABAh response (-69 mV), and was significantly different from the reversal potential of the GABAd response (-56 mV). The GABAd response was more sensitive to enhancement by pentobarbital and more readily antagonized by both bicuculline and picrotoxin than the GABAh response. Recording in bicarbonate-free buffer changed the reversal potential of the GABAd response significantly, but had no effect on the GABAh response. In contrast, superfusion with ethanol significantly enhanced the GABAh response, while having no effect on the GABAd component. Although a localized collapse of the Cl- gradient, which has been proposed to underlie the GABAd response, could explain the greater sensitivity of the GABAd response to pentobarbital and the GABAA antagonists, this could not account for the greater sensitivity of the GABAh response to ethanol. Differences in GABAA receptor subunit composition may result in the expression of dendritic and somatic GABAA receptors that have different kinetics, reversal potentials, and sensitivity to pharmacological agents, including ethanol.

Electrophysiological Analysis of G Protein–Coupled Receptors in Mammalian Neurons

This unit describes general techniques that are useful for recording electrophysiological responses that are mediated via the activation of G-protein coupled receptors (GPCRs). It includes a brief description of preparations, but focuses primarily on experiments using hippocampal brain slice preparations. Techniques for the preparation of brain slices, electrodes, filling solutions, and the recording protocols that are suitable for assessing the activity of GPCRs using electrophysiological techniques are summarized, and various protocols for the activation of these receptors are discussed.

In vitro electrophysiological analysis of mature rat hippocampal transplants in oculo

We have investigated the maturation of isolated rat hippocampus grafted into the anterior chamber of the eye. Electrophysiological responses from transplants were compared to those recorded from the in vitro hippocampal slice preparation. Intracellular recording demonstrated that the passive membrane characteristics of intraocular hippocampal neurons were similar to those of the CA1 pyramidal cells in the in vitro slice preparation. However, the slow after-hyperpolarization which normally follows depolarization-induced action potentials was reduced or completely absent in the intraocular transplants, and the excitatory postsynaptic potential (EPSP) evoked by local stimulation was prolonged. The duration of the EPSP was reduced by perfusion with D-aminophosphonovaleric acid (2.5-50 microM), an N-methyl-D-aspartate receptor antagonist. Normal levels of glutamate decarboxylase (a marker for gamma-aminobutyric acidergic neurons) were found in the transplants, and responses to adenosine, bicuculline, and norepinephrine were similar in the in oculo transplants and in vitro slices. The data suggest that although many properties of hippocampal neurons are intrinsically determined, other aspects of the physiology of mature hippocampus either fail to develop, or develop abnormally in the absence of external inputs in oculo.