The regulation of glutamatergic neurotransmission may also contribute to the antinociceptive activity of cannabinoid agonists. In the dorsal horn of the spinal cord, these agents suppress glutamate release from primary sensory afferents. Whole cell patch-clamp recordings in substantia gelatinosa neurons have indeed demonstrated that cannabinoid agonists reduce both the amplitude and frequency of spontaneous EPSCs . The frequency, but not the amplitude, of miniature EPSCs was diminished, indicating a presynaptic effect. The presumptive target sites of these effects are the axon terminals of afferent sensory fibers, since evoked EPSCs are also significantly decreased by cannabinoid agonists upon stimulation of the neighboring dorsal root ganglion. The stimulation protocol used in this study indicated that mainly A_x0005_ – and C-fibers were affected, which was also confirmed by using the vanilloid agonist capsaicin . These results parallel anatomical evidence that dorsal root ganglion neurons express CB1 receptors , and cannabinoid binding sites are reduced after dorsal rhizotomy or neonatal capsaicin treatment, although only 16% of the total CB1 receptor population was estimated to be located on C-fibers . Along with the spinal cord, glutamatergic neurotransmission is also affected in neurons of the PAG , which may also contribute to the role of cannabinoids in alleviating pain sensation. Interestingly, however, it seems that the inhibitory effect of cannabinoids on glutamate release cannot be extended to all regions involved in antinociceptive activity of cannabinoids. Remarkably,cannabis drying racks while GABAergic neurotransmission was massively inhibited by cannabinoids in the trigeminal nucleus caudalis, the evoked EPSCs upon stimulation of the trigeminal tract remained unaffected .
However, the trigeminal tract contains a mixed population of glutamatergic axons with different conduction velocities and activation thresholds. The C-fibers exhibit a higher activation threshold, and thus the effect of cannabinoids on a selected small population of fibers may be masked by the use of different stimulation protocols .As observed throughout the brain, CB1-bearing terminals in the spinal cord contain modulatory neuropeptides in addition to fast-acting amino acid neurotransmitters. Two neuropeptides, substance P and CGRP, are coexpressed with CB1 receptors in dorsal root ganglia . Accordingly, low doses of the endocannabinoid anandamide inhibit capsaicin-evoked CGRP release from both central and peripheral axon terminals of primary sensory neurons in the dorsal horn of the spinal cord, as well as in hindpaw skin . In addition, at low concentrations, anandamide also inhibits SP and CGRP release elicited by electrical field stimulation , an effect that probably results from the activation of CB1 receptors and may contribute to the analgesic and anti-inflammatory properties of this lipid mediator . At high concentrations, which are unlikely to be attained in vivo, anandamide activates capsaicin-sensitive VR1 receptors, thereby stimulating SP and CGRP release.The physiological significance of these findings, if any, is unknown at present.Cannabinoids can evoke physiological responses, which may not be mediated by presynaptic cannabinoid receptors. Recent reports indicate that both endocannabinoids and synthetic cannabinoid agonists modify the ex-citability of neurons via regulation of distinct potassium conductances present on the extrasynaptic dendritic surface of neurons . Within the synapse, the modulation of excitatory postsynaptic responses mediated by NMDA receptors was also reported . In addition, cannabinoids are also able to induce or suppress gene expression patterns by activating signal transduction pathways likely to occur in the postsynaptic domain of neurons .
However, in most cases, the molecular substrates of these effects have not been unequivocally identified. Certain cannabinoid compounds were shown to activate ion channels and receptors other than CB1 receptors . In addition, although many of the cannabinoid effects mentioned above were blocked by the CB1 antagonist SR141716A, experiments with CB1 /_x0005_ mice demonstrate that this antagonist also recognizes other CB1-like receptors . Further studies on genetically modified animals and novel, more selective pharmacological tools are thus needed to dissect all the molecular components of cannabinoid neuromodulation. In contrast to the well-established evidence of presynaptic CB1 receptors in various brain regions and on the axon terminals of a number of distinct cell types, the presence of functional CB1 receptors on the plasma membrane of the dendritic tree or somata of neurons requires more solid evidence than available at present. Although postsynaptic CB1 receptors have been suggested to exist , published data show a clear mismatch between the subcellular localization of the protein epitope used to generate the antibody and the distribution pattern of immuno labeling. The antibody used in these studies was generated against the NH2 terminus of the CB1 receptor protein , which is expected to be situated on the outer surface of the plasma membrane. In contrast, in these studies dendritic CB1 labeling is invariably found inside cells and is often distant from the plasma membrane. This pattern might represent a labeling artifact, common in immunogold and immunoperoxidase staining, or may be biologically relevant. In some cases, dendritic CB1 immunolabeling is clearly associated with intracellular organelles participating in the processing or degradation of proteins, such as the rough endoplasmic reticulum, the Golgi apparatus, or multivesicular bodies . Since these intracellular organelles usually intrude into the cytoplasm of proximal dendrites, in single electron microscopic sections they appear to be located within dendritic segments.
Nevertheless, these segments belong functionally to the somatic region, because they compose a continuous network within these structures. In our view, the most likely explanation for this labeling pattern is that the antibody also recognizes the freshly synthesized or degraded CB1 protein. In support of this idea, correlated light and electron microscopy using high-resolution immunogold technique provide clear-cut evidence that the CB1 immunostaining visualizing cell bodies and proximal dendrites of interneurons at the light microscopical level is always associated with intracellular organelles, but never with the somatic or dendritic plasma membrane . Moreover, the antibody recognizing the NH2 terminus of the CB1 receptor selectively labels the axon terminals of these interneurons, and the gold particles are found exclusively on the outer surface of the plasma membrane, demonstrating the availability of the NH2-terminal epitope for this antibody in conventional electron microscopic preparations . Thus, in contrast to presynaptic CB1 receptors,cannabis grow tray establishing the presence of such receptors on the plasma membrane of the dendritic tree or somata of neurons will require further experimentation.In the year 2001, we witnessed the merger of two independent lines of research, namely, decades of investigations into the cellular and network effects of exogenous cannabinoids, and studies on the characteristics of DSI, a form of retrograde synaptic signaling. Wilson and Nicoll and Ohno-Shosaku et al. provided the missing link between these two lines by demonstrating that an as-yet-unidentified endocannabinoid substance mediates DSI. If we want to evaluate the studies that led to the present understanding of endocannabinoid functions, we should follow the milestones of research not only in the field of cannabinoid pharmacology, but also the sequence of discoveries that led to the establishment of the phenomenon, as well as the pharmacology and physiology of DSI, namely, the work that was initiated by the groups of Alger and Marty in the early 1990s . Thus this section will synthesize the findings deriving from these two roots of research with the aim to better understand the functional roles of endocannabinoids at the synaptic and network levels.It has been known for decades that cannabinoids have a profound influence on learning and memory . This may be related 1) to the impairment of long-term potentiation that is generally believed to be linked to learning-associated synaptic plasticity of glutamatergic connections, 2) to a disturbance of fast and slow oscillations maintained by GABAergic interneurons that secure the necessary synchrony in the discharges of connected neurons, or 3) to alterations in the activity or release properties of monoaminergic and cholinergic subcortical pathways known to influence cortical plasticity and activity states.
Thus the major questions here concern the brain region and transmitter system involved, as well as the network mechanisms underlying the cannabinoid effects. In addition to showing an intense CB1 receptor binding, the hippocampus is known to be a crucial area involved in learning and memory. LTP, as well as fast and slow oscillations, has been investigated most extensively and reproducibly in this brain region, and the underlying synaptic connectivity is relatively well understood. These features together provided sufficient reason to focus the majority of cannabinoid electrophysiology, and a large part of this section of the present review, on the hippocampus. On the other hand, the cerebellum and the basal ganglia are also extensively studied in cannabinoid physiology due to the well-known behavioral effects associated with these regions , as well as to the very high density of cannabinoid binding sites . In addition, the cerebellum was one of the areas where DSI was discovered . Cannabinoid effects in these two brain regions have been discussed at the cellular level in sections III and IV; here we only focus on implications for DSI/DSE and, whenever data are available, possible network mechanisms. Glutamate is the major mediator of intracellularly recorded as well as field EPSPs in the hippocampus, and it is the transmitter at synapses that are best known to show long-term plastic changes in strength. In addition, the laminar distribution of CB1 receptor binding in the hippocampus overlaps with glutamatergic pathways, which together explains why this transmitter has been investigated most extensively. On the other hand, both fast and slow oscillations rely on local GABAergic interneurons in the hippocampus , and in addition, GABA is by far the most dominant neurotransmitter in the cerebellum and basal ganglia as well, providing ample reason for focusing studies also on this transmitter. In addition to influencing learning and memory, cannabinoids have a profound effect on mood, emotions, and motivation, which are known to involve subcortical monoaminergic pathways. Therefore, effects of cannabinoids on dopaminergic, cholinergic, serotonergic, and noradrenergic transmission have also been extensively studied, mostly in the basal ganglia, and to some extent also in the hippocampus, amygdala, and neocortex .In general, any drug actions on field EPSPs, population spikes, and paired-pulse synaptic plasticity are difficult to interpret, since several mechanisms may underlie any observed changes. These mechanisms should be studied by intracellular recordings from single cells or connected cell pairs, and parallel population data should be provided. Such combined studies are rather rare in the cannabinoid field; therefore, we chose to present the data from the literature without necessarily attempting to provide an explanation for the mechanism of cannabinoid actions, and for the conflicting data. Many of the conflicting results may be due to the dual cannabinoid actions on CB1 and on the new cannabinoid-sensitive receptor that is present on glutamatergic axons in the hippocampus , which can be influenced by several of the agonists and antagonists that are extensively used today as “selective” CB1 ligands . Some controversial interpretations of earlier studies may result from the shortage of data on the pre- or postsynaptic localization of the cannabinoid receptor. Our interpretation of these earlier results rests on the recent knowledge that these receptors are mostly, if not exclusively, presynaptic, as reviewed in section IV. One of the earliest electrophyiological studies using cannabinoid agoinsts found that cannabinoids suppressed sensory-evoked or spontaneous firing of dentate granule cells and elicited characteristic changes in evoked potential waveform . In the hippocampus, Wilkison and Pontzer showed that CB1 agonists and antagonists had negligible effects on field EPSPs and population spikes, whereas in some other studies cannabinoids were shown to have a dose-dependent biphasic effect on evoked population spikes. At low doses, delta-9-THC augments evoked field EPSPs as well as orthodromically or antidromically evoked population spikes, whereas at higher doses the responses are depressed . In recent studies, the endogenous ligand anandamide was shown to decrease the slope of Schaffer collateral-evoked field EPSPs, as well as the amplitude of population spikes in the CA1 region at relatively low and high concentrations as well . The antagonist SR141716 prevented the effect of anandamide and when applied on its own induced a small increase in population spike amplitude. This suggests that endogenously released cannabinoids may be capable of inhibiting glutamate release. In contrast, another endocannabinoid, 2-AG, had no effect on the slope of evoked field EPSPs in CA1 , implying that the endogenous cannabinoid action observed by Ameri et al. using SR141716A is likely exerted by anandamide alone.