Characterization of the kinetics and distribution of N-arachidonylethanolamine (anandamide) hydrolysis by rat brain.

Arachidonoylethanolamide or 'anandamide' is a naturally occurring derivative of arachidonic acid that has been shown to activate cannabinoid receptors in the brain. Its metabolic inactivation by brain tissue has been investigated. Anandamide is hydrolyzed by the membrane fraction of rat brain homogenate to arachidonic acid and ethanolamine. The hydrolysis is temperature and pH- dependent (pH maximum at 8.5) and abolished by boiling. Anandamide hydrolysis is protein dependent in the range of 25-100 micrograms protein/ml; does not require calcium and is inhibited by phenylmethylsulfonylfluoride, diisopropylfluorophosphate, thimerosal and arachidonic acid. Hydrolysis of 10 microM anandamide by brain membranes follows first order kinetics; at 30 degrees C, the rate constant for anandamide catabolism is 0.34 min-1 mg protein-1. The Km for anandamide hydrolysis is 3.4 microM, and the Vmax is 2.2 nmol/min per mg protein. Hydrolysis occurs in all subcellular fractions except cytosol with the highest specific activity in myelin and microsomes. The distribution of anandamide hydrolytic activity correlates with the distribution of cannabinoid receptor-binding sites; the hippocampus, cerebellum and cerebral cortex exhibit the highest metabolic activity, while activity is lowest in the striatum, brain stem and white matter.

Slowing of cortical somatosensory evoked activity by delta 9-tetrahydrocannabinol and dimethylheptylpyran in alpha-chloralose-anesthetized cats.

Somatosensory and visual evoked potentials in the classical afferent primary pathways in cats anesthetized with alpha-chloralose were studied in order to characterize the effects of delta 9-tetrahydrocannabinol (THC) and the synthetic analog, dimethylheptylpyran (DMHP) on central sensory processing. THC and DMHP slowed primary cortical responses to radial nerve or ventralis posterolateralis (VPL) stimulation in a dose dependent manner. THC did not alter VPL activity evoked by radial nerve stimulation. Amplitude of the primary somatic response was depressed at the 4 mg/kg dose. Responses of visual cortex and lateral geniculate nucleus, evoked by stimulation of optic chiasm, were unchanged at the doses studied. However, the nonmodality-specific response of the anterior marginal association cortex to stimulation of either modality was depressed by THC. Data suggest the association cortex which exhibits sensory convergence was most sensitive to the effects of THC. Findings were consistent with a thalamocortical site of action for THC. DMHP produced similar effects and was more potent.

Estimation of thresholds for evoked potentials using a laboratory computer.

To study drug effects on bioelectric activity evoked by near-threshold stimulation, we have developed a computerized threshold estimation technique. The procedure utilizes a laboratory computer (PDP 11/34) equipped with a standard laboratory interface with A/D and D/A capabilities. Program-controlled voltage pulses, delivered via D/A, are interfaced through a constant current stimulus isolation unit for electrical stimulation. One channel of activity is digitized at a user-defined rate and the signal is searched for a response. Responses were generally defined by a feature of the signal which was greater than a multiple of its standard deviation in the subthreshold signal. Using some a priori knowledge of the expected response and user-defined processing options, the program determines threshold on-line without pretrial test data. Program operation is illustrated with sensory evoked potentials in acutely prepared cats and hippocampal evoked potentials in chronically implanted rats.

Separation of components of hippocampal field potentials by digital filtering on a PC-based microcomputer.

A versatile digital filtering system is described which enables separation of wave components of evoked potentials based on their frequencies. The utility of this technique is exemplified by application of digital filtering to hippocampal field potentials. Separation of population spikes from lower frequency synaptic potentials provides less ambiguous interpretation of the compound slope of the field potential during the first 5 milliseconds of the response. The entire procedure of stimulation, data acquisition, and digital filtering was implemented on a PC-compatible computer.

The actions of THC on the intact hippocampus: a comparison of dentate and CA1 responses.

The effects of delta-9-tetrahydrocannabinol were compared on two afferent pathways of the rat hippocampal formation in urethane-anesthetized and chronically implanted awake animals. In urethane-anesthetized rats, THC produced a shift to the right in the input/output relationship without altering the relation between the measures of synaptic wave and the population spike of the CA1 response to contralateral CA3 stimulation. In contrast, the dentate response to perforant path stimulation was not altered by THC. Awake rats exhibited a similar response to THC, however, the CA1 response was less sensitive to THC and there was no change in the maximal response as there was in the anesthetized rat. Paired pulse studies demonstrated that there was a significant increase in paired pulse inhibition at 8 mg/kg in the CA1 region. There was no significant alteration of the dentate response to perforant path stimulation in the awake rat. These results suggest that alterations in limbic electrophysiology induced by THC may be mediated by structures other than the hippocampus.

Effects of intraocular mescaline and LSD on visual-evoked responses in the rat.

The effects of mescaline and LSD on the flash-evoked cortical potential (FEP) were determined in unrestrained rats with chronically-implanted electrodes. Systemic administration of mescaline or LSD significantly attenuated the primary component of the FEP at three stimulus intensities with the greatest effect observed 60-90 minutes following drug administration. The magnitude and specificity of the effects of these agents on the primary response suggest that they produce deficits in conduction through the retino-geniculato-cortical system. The serotonin receptor antagonists, cyproheptadine and methysergide, antagonized the mescaline-induced depression of the FEP in accordance with neurochemical and behavioral evidence that mescaline acts as a partial agonist on serotonin receptors. Topical or intraocular administration of atropine antagonized the actions of systemically-administered mescaline. In addition, intraocular administration of mescaline or LSD attenuated the FEP indicative of an action of these hallucinogens on visual processing in the retina which is modulated by muscarinic receptor activity.

Inhibition of heterosensory thalamocortical evoked potentials by delta-9-tetrahydrocannabinol.

The effects of delta-9-tetrahydrocannabinol on sensory activity in the thalamic intralaminar nuclei, centralis lateralis and the mesencephalic reticular formation were compared with the effects on cortical association or heterosensory systems in alpha-chloralose-anesthetized cats. The drug depressed the anterior marginal responses to multiple-modality sensory stimulation 30 min after administration of 2 mg/kg. Posterior suprasylvian responses were not significantly depressed except during 15 min postadministration. The drug did not depress sensory responses at the centralis lateralis or the mesencephalic reticular formation. Cortical responses evoked by stimulation of the latter were globally depressed by the drug. In contrast, delta-9-tetrahydrocannabinol depressed the anterior marginal response to centralis lateralis stimulation and not the posterior suprasylvian response similarly to the responses to sensory stimulation. The drug was without effect on caudate nucleus responses to stimulation of the mesencephalic reticular formation or centralis lateralis. These data demonstrate that, unlike pentobarbital, delta-9-tetrahydrocannabinol maintains heterosensory afferent activity to mesencephalic and thalamic sites and that its unique properties appear to be due, in part, to the selective disruption of heterosensory thalamocortical function.

Differential effects of lysergic acid diethylamide, methysergide, and cyproheptadine on modality-specific and nonspecific sensory evoked potentials.

The effects of lysergic acid diethylamide (LSD), methysergide, and cyproheptadine on activity in classical primary pathways of the visual and somatosensory systems were compared with their effects on activity in sensory convergent (association) regions in alpha-chloralose-anesthetized cats. Those effects were blocked by cyproheptadine whereas methysergide potentiated the actions of LSD on visual primary activity. In contrast, LSD depressed the primary somatic pathway, at small doses (25 to 50 micrograms/kg) and facilitated the response at larger doses (200 micrograms/kg). Cyproheptadine and methysergide did not agonize these actions of LSD. The anterior marginal cortex, nucleus central median-parafascicularis, nucleus lateral posterior, and the superior colliculus, all sites of heterosensory convergence, were depressed by LSD. The depression of responses at heterosensory sites by LSD was blocked by cyproheptadine. Methysergide potentiated the LSD-induced depression of visual-evoked activity but not somatosensory activity. These results suggest that LSD depresses sensory activity in regions which integrate multiple sensory modalities independently of actions on sensory-specific pathways. These effects appear to involve a cyproheptadine-sensitive system.

A microcomputer-based package for determination of regional and global cardiac function and coronary hemodynamics.

A multichannel data acquisition package for reduction of systemic and coronary hemodynamic data that utilizes a personal microcomputer is described. The system provides a printout of heart rate, maximal rate of left ventricular pressure development (+dP/dt), maximal rate of ventricular relaxation (-dP/dt), absolute and normalized myocardial segment lengths, the degree of shortening of the segment over the cardiac cycle, systolic, diastolic, and mean coronary blood flow velocities, left ventricular systolic and end diastolic pressures, and systolic, diastolic, and mean aortic blood pressure from six channels of input data. Obtained values are precisely linked to the cardiac cycle. To illustrate the output of this system, data obtained by reading strip-chart records are compared to microcomputer-derived values in conscious, instrumented dogs.

Alteration of electrical correlates of sensory processing by tetrahydrocannabinol.

The action of tetrahydrocannabinol on the function of four multimodality responsive neocortical areas was assessed in the cat. Cats, anesthetized with alpha-chloralose, were subjected to auditory, somatosensory, and two types of visual stimulation. Simultaneous evoked field potentials and multiple-unit activity were recorded. The drug produced a significant reduction in evoked field responses at the three most rostral cortical areas, whereas responses at the posterior suprasylvian gyrus were spared. There were no significant differences between sensory modalities. In addition to being the first pharmacological study to demonstrate differential drug sensitivity between these polysensory areas, these studies localized an effect of tetrahydrocannabinol. Sensory evoked multiple-unit activity showed a pattern of change after THC consistent with a reduction in activity of pyramidal output cells and a concomitant increase in interneuron activity. The action of tetrahydrocannabinol on polysensory systems is important in attention and orientation.