Cannabis Extract CT-921 Has a High Efficacy-Adverse Effect Profile in a Neuropathic Pain Model.

BACKGROUND: Legalization of cannabis encourages the development of specific cultivars to treat disease such as neuropathic pain. Because of the large number of cultivars, it is necessary to prioritize extracts before proceeding to clinical trials. PURPOSE: To compare extracts of two unique cannabis cultivars (CT-921, CT-928) for treatment of neuropathic pain induced by constriction of sciatic nerve in mice and to illustrate the use of this animal model to set priority for future trials. METHODS: Pain severity was measured by threshold force causing paw withdrawal. Dose-response relationships and time course were determined for intravenously injected extracts of cultivars and vehicle. The doses for allodynia relief were correlated with decreased respiratory rate, temperature and behavioral changes. RESULTS: Effective analgesic dose for 50 and 95% (ED50An and ED95An) was 15, and 29 mg/kg for CT-921 and 0.9 and 4.7 for CT-928. At ED50An, for both extracts, the duration was 120 min. At ED95An, administration of CT-928 significantly decreased respiratory rate while CT-921 did not. CT-928 decreased temperature more than CT-921. CT-928 produced frantic hyperactivity not seen with CT-921. At equivalent analgesic doses, THC was much less in CT-921 than in CT-928 suggesting interactions with components other than THC influenced the analgesia. At equivalent analgesic doses, efficacy-to-adverse effect profile for CT-928 was worse than for CT-921. CONCLUSION: Both extracts relieved neuropathic pain; however, CT-921 had a better efficacy-to-adverse effect profile, a rational basis for prioritizing cultivars for future clinical evaluation.

Repeated Testing With the Hypertonic Saline Assay in Mice for Screening of Analgesic Activity.

BACKGROUND: In vivo animal assays are a cornerstone of preclinical pain research. An optimal stimulus for determining the activity of potential analgesics would produce responses of a consistent magnitude on repeated testing. Intraplantar (i.pl.) injection of hypertonic saline (HS) in mice produces robust nociceptive responses to different analgesics, without evidence of tissue damage. Here, we investigated whether the nociceptive response is changed by repeating the injection at different times and sites in a mouse and whether it is attenuated by morphine. METHODS: We conducted randomized and blinded experiments to assess responses to repeated i.pl. 10% HS in female CD-1 mice. An injection of HS was followed by a second injection into the same hind paw at 4 hours, 24 hours, or 7 days. A separate group of mice each received i.pl. injections at 5, 10, and 15 days. In 2 independent experiments, 30 minutes after initial HS injections in the ipsilateral hind paw, mice received HS injection into the contralateral hind paw or ipsilateral forepaw. The ability of morphine to block the nociceptive responses was examined by injecting morphine at 5-day intervals. RESULTS: Repeated injection of HS did not alter the responses at 4 hours (84 vs 75 seconds; mean difference [95% CI], -9 [-40 to 23]; P = .6), 24 hours (122 vs 113 seconds; -6 [-24 to 12]; P = .5), or 7 days (112 vs 113 seconds; -0.3 [-12 to 11]; P = .95) or at multiple injections (day 0, 122 seconds vs day 5, 121 seconds; -0.3 [-28 to 27], P > .99; day 10, 118 seconds; 2.5 [-36 to 41], P = .99; day 15, 119 seconds; 2 [-36 to 38], P = .99). A previous hind paw injection did not change the responses of the contralateral hind paw (right, 93 seconds versus left, 96 seconds; -3 [-20 to 13], P = .7) or of the ipsilateral forepaw (forepaw after HS, 146 seconds versus forepaw after 0.9% saline, 149 seconds; -3 [-28 to 22], P = .8). Morphine dose-dependently attenuated HS responses (control, 94 seconds vs 4 mg/kg, 66 seconds; 29 [-7 to 64], P = .12; vs 10 mg/kg, 27 seconds; 67 [44-90], P < .0001; 4 vs 10 mg/kg, 67 [44-90], P = .03). CONCLUSIONS: The repetition of i.pl. HS produces consistent reproducible responses without tissue damage. This results in efficient, rapid detection of analgesic activity, reducing the number of animals required.

Fluctuation analysis of tetanic rundown (short-term depression) at a corticothalamic synapse.

Hypothetical scenarios for "tetanic rundown" ("short-term depression") of synaptic signals evoked by stimulus trains differ in evolution of quantal amplitude (Q) and covariances between signals. With corticothalamic excitatory postsynaptic currents (EPSCs) evoked by 2.5- to 20-Hz trains, we found Q (estimated using various corrections of variance/mean ratios) to be unchanged during rundown and close to the size of stimulus-evoked "miniatures". Except for covariances, results were compatible with a depletion model, according to which incomplete "refill" after probabilistic quantal release entails release-site "emptying". For five neurons with 20 train repetitions at each frequency, there was little between-neuron variation of rundown; pool-refill rate increased with stimulus frequency and evolved during rundown. Covariances did not fit the depletion model or theoretical alternatives, being excessively negative for adjacent EPSCs early in trains, absent at equilibrium, and anomalously positive for some nonadjacent EPSCs. The anomalous covariances were unaltered during pharmacological blockade of receptor desensitization and saturation. These findings suggest that pool-refill rate and release probability at each release site are continually modulated by antecedent outputs in its neighborhood, possibly via feedback mechanisms. In all data sets, sampling errors for between-train variances were much less than theoretical, warranting reconsideration of the probabilistic nature of quantal transmitter release.

Barbiturate activation and modulation of GABA(A) receptors in neocortex.

We determined if anesthetic and anti-epileptic barbiturates inhibit neurons by different mechanisms. Current- and voltage-clamp recordings were made from somatosensory neurons of neocortex and some thalamocortical neurons in coronal brain slices of rats. We compared effects of pentobarbital, amobarbital, and phenobarbital on inhibitory postsynaptic currents (IPSCs) mediated by gamma-aminobutyric acid (GABA), input conductance, and evoked action potential firing. In neocortex, pentobarbital (EC(50)=41 microM) and amobarbital (EC(50)=103 microM) increased the decay time constant of GABA(A)ergic IPSCs. At higher concentrations, pentobarbital and amobarbital shunted firing by increasing input conductance through agonism at GABA(A) receptors. At anti-epileptic concentrations, phenobarbital increased the IPSC decay time constant (EC(50)=144 microM), and shunted firing by agonism at GABA(A) receptors (EC(50)=133 microM). In thalamocortical neurons, similar concentrations of phenobarbital had negligible effects on GABA(A)ergic IPSCs, conductance, and firing. In contrast to their thalamic actions, barbiturates inhibit neocortical neurons mostly through GABA receptors. Neocortical enhancement of inhibition by pentobarbital and amobarbital, combined with actions on thalamocortical neurons, may contribute to redundant mechanisms of anesthesia. The ability of phenobarbital at anti-epileptic concentrations to inhibit neocortical firing by direct activation and modulation of GABA(A) receptors relates to its specialized therapeutic effects.

Distinctive glycinergic currents with fast and slow kinetics in thalamus.

We examined functional properties of inhibitory postsynaptic currents (IPSCs) evoked by medial lemniscal stimulation, spontaneous IPSCs (sIPSCs), and single-channel, extrasynaptic currents evoked by glycine receptor agonists or gamma-aminobutyric acid (GABA) in rat ventrobasal thalamus. We identified synaptic currents by reversal at E(Cl) and sensitivity to elimination by strychnine, GABA(A) antagonists, or combined application. Glycinergic IPSCs featured short (about 12 ms) and long (about 80 ms) decay time constants. These fast and slow IPSCs occurred separately with monoexponential decays, or together with biexponential decay kinetics. Glycinergic sIPSCs decayed monoexponentially with time constants, matching fast and slow IPSCs. These findings were consistent with synaptic responses generated by two populations of glycine receptors, localized under different nerve terminals. Glycine, taurine, or beta-alanine applied to excised membrane patches evoked short- and long-duration current bursts. Extrasynaptic burst durations resembled fast and slow IPSC time constants. The single, intermediate time constant (about 22 ms) of GABA(A)ergic IPSCs cotransmitted with glycinergic IPSCs approximated the burst duration of extrasynaptic GABA(A) channels. We noted differences between synaptic and extrasynaptic receptors. Endogenously activated glycine and GABA(A) receptor channels had higher Cl- permeability than that of their extrasynaptic counterparts. The beta-amino acids activated long-duration bursts at extrasynaptic glycine receptors, consistent with a role in detection of ambient taurine or beta-alanine. Heterogeneous kinetics and permeabilities implicate molecular and functional diversity in thalamic glycine receptors. Fast, intermediate, and slow inhibitory postsynaptic potential decays, mostly attributed to cotransmission by glycinergic and GABAergic pathways, allow for discriminative modulation and integration with voltage-dependent currents in ventrobasal neurons.

Glycinergic inhibition in thalamus revealed by synaptic receptor blockade.

Using juvenile rat brain slices, we examined the possibility that strychnine-sensitive receptors for glycine-like amino acids contributed to synaptic inhibition in ventrobasal thalamus, where gamma-aminobutyrate (GABA) is the prevalent inhibitory transmitter. Ventrobasal nuclei showed staining for antibodies against alpha1 and alpha2 subunits of the glycine receptor. Exogenously applied glycine, taurine and beta-alanine increased membrane conductance, effects antagonized by strychnine, indicative of functional glycine receptors. Using glutamate receptor antagonists, we isolated inhibitory postsynaptic potentials and currents (IPSPs and IPSCs) evoked by high-threshold stimulation of medial lemniscus. Like the responses to glycine agonists, these synaptic responses reversed near E(Cl). In comparative tests with GABA receptor antagonists, strychnine attenuated inhibition in a majority of neurons, but did not alter slow, GABA(B) inhibition. For complete blockade, the majority of fast IPSPs required co-application of strychnine with bicuculline or gabazine, GABA(A) receptor antagonists. Strychnine acting with an IC50 approximately = 33 nM, eliminated residual fast inhibition during selective GABA(A) receptor blockade with gabazine. The latency of onset for IPSPs was compatible with polysynaptic pathways or prolonged axonal propagation time. Strychnine lacked effects on monosynaptic, GABAergic IPSPs from zona incerta. The specific actions of strychnine implicated a glycine receptor contribution to fast inhibition in somatosensory thalamus.

Membrane resonance and stochastic resonance modulate firing patterns of thalamocortical neurons.

We examined the interactions of subthreshold membrane resonance and stochastic resonance using whole-cell patch clamp recordings in thalamocortical neurons of rat brain slices, as well as with a Hodgkin-Huxley-type mathematical model of thalamocortical neurons. The neurons exhibited the subthreshold resonance when stimulated with small amplitude sine wave currents of varying frequency, and stochastic resonance when noise was added to sine wave inputs. Stochastic resonance was manifest as a maximum in signal-to-noise ratio of output response to subthreshold periodic input combined with noise. Stochastic resonance in conjunction with subthreshold resonance resulted in action potential patterns that showed frequency selectivity for periodic inputs. Stochastic resonance was maximal near subthreshold resonance frequency and a high noise level was required for detection of high frequency signals. We speculate that combined membrane and stochastic resonances have physiological utility in coupling synaptic activity to preferred firing frequency and in network synchronization under noise.

Resonances and noise in a stochastic Hindmarsh-Rose model of thalamic neurons.

Thalamic neurons exhibit subthreshold resonance when stimulated with small sine wave signals of varying frequency and stochastic resonance when noise is added to these signals. We study a stochastic Hindmarsh-Rose model using Monte-Carlo simulations to investigate how noise, in conjunction with subthreshold resonance, leads to a preferred frequency in the firing pattern. The resulting stochastic resonance (SR) exhibits a preferred firing frequency that is approximately exponential in its dependence on the noise amplitude. In similar experiments, frequency dependent SR is found in the reliability of detection of alpha-function inputs under noise, which are more realistic inputs for neurons. A mathematical analysis of the equations reveals that the frequency preference arises from the dynamics of the slow variable. Noise can then transfer the resonance over the firing threshold because of the proximity of the fast subsystem to a Hopf bifurcation point. Our results may have implications for the behavior of thalamic neurons in a network, with noise switching the membrane potential between different resonance modes.

Spermine modulates neuronal excitability and NMDA receptors in juvenile gerbil auditory thalamus.

Medial geniculate body (MGB) neurons process synaptic inputs from auditory cortex. Corticothalamic stimulation evokes glutamatergic excitatory postsynaptic potentials (EPSPs) that vary markedly in amplitude and duration during development. The EPSP decay phase is prolonged during second postnatal week but then shortens, significantly, until adulthood. The EPSP prolongation depends on spermine interactions with a polyamine-sensitive site on receptors for N-methyl-D-aspartate (NMDA). We examined effects of spermine application on EPSPs, firing modes, and membrane properties in gerbil MGB neurons during the P14 period of highest polyamine sensitivity. Spermine slowed EPSP decay and promoted firing on EPSPs, without changing passive membrane properties. Spermine increased membrane rectification on depolarization, which is mediated by tetrodotoxin (TTX)-sensitive, persistent Na(+) conductance. As a result, spermine lowered threshold and increased tonic firing evoked with current injection by up to approximately 150%. These effects were concentration-dependent (ED(50)=100 microM), reversible, and eliminated by NMDA receptor antagonist, 2-amino-5-phosphonovalerate (APV). In contrast, spermine increased dV/dt of the low threshold Ca(2+) spike (LTS) and burst firing, evoked from hyperpolarized potentials. LTS enhancement was greater at -55 mV than at hyperpolarized potentials and did not result from persistent Na(+) conductance or glutamate receptor mechanisms. In summary, spermine increased excitability by modulating NMDA receptors in juvenile gerbil neurons.

Pentobarbital depressant effects are independent of GABA receptors in auditory thalamic neurons.

Pentobarbital, a general anesthetic, has received extensive study for its ability to potentiate inhibition at GABA(A) subtype of receptors for GABA. Using whole cell current-clamp techniques and bath applications, we determined the effects of pentobarbital and GABA receptor antagonists on the membrane properties and tonic or burst firing of medial geniculate neurons in thalamic slices. Pentobarbital (0.01-200 microM) induced depressant effects in 50 of 66 neurons (76%). Pentobarbital hyperpolarized neurons by a mean of 3 mV and decreased the number of action potentials in tonic firing, evoked by current pulse injection from near the resting potential. Pentobarbital also decreased burst firing or low threshold Ca(2+)-spikes, evoked by current pulse injection into neurons at potentials hyperpolarized from rest. The blockade of tonic and burst firing, as well as low threshold Ca(2+)-spikes, was surmountable by increasing the amplitude of input current. The GABA(A) receptor antagonists, bicuculline (100 microM) and picrotoxinin (50-100 microM), did not block the depressant effects of pentobarbital (10 microM). The GABA(B) receptor antagonist, saclofen (200 microM), and GABA(C) receptor antagonist, (1,2,3,6-tetrahydropyridine-4-yl)methylphosphinate (10-50 microM), did not significantly alter the depressant effects. Pentobarbital produced excitatory effects (0.1-50 microM) on 11 neurons (17%) but had no effects on 5 neurons (7%). The excitation consisted of approximately 3 mV depolarization, increased tonic and burst firing and the rate of rise and amplitude of low threshold Ca(2+) spikes. These effects were associated with a increase in input resistance. In contrast, the depressant effects of pentobarbital correlated to a decreased input resistance measured with hyperpolarizing current pulse injection (IC(50) = 7.8 microM). Pentobarbital reduced Na(+)-dependent rectification on depolarization and lowered the slope resistance over a wide voltage range. Tetrodotoxin eliminated both Na(+)-dependent rectification and the pentobarbital-induced decrease in membrane resistance at depolarized voltages in two-thirds of the neurons. The pentobarbital-induced decrease in membrane resistance at voltages hyperpolarized from rest was not evident during co-application with Cs(+), known to block the hyperpolarization-activated rectifiers. In summary, the pentobarbital acted at low concentrations to depress thalamocortical neurons. The depression resulted from decreased rectification on depolarization, which no longer boosted potentials over threshold, and an increased conductance that shunted spike generation. The depressant effects of pentobarbital did not involve known types of GABA receptor interactions.