Such an “inverted U” dose-response relationship warrants further investigation but may be explained by the activation of different cannabinoid receptor types, likely possessing distinct agonist sensitivity , or by the dose-dependent engagement of excitatory or inhibitory afferent pathways of the basal forebrain, which may enhance or reduce the intrinsic activity of cholinergic neurons. In any case, the predominant effect of presynaptic CB1 receptors present on cholinergic axon terminals within the cortex is likely to be the inhibition of acetylcholine release, although such an effect alone may not entirely explain cannabinoid actions on cognition .Along with cholinergic fibers, ascending noradrenergic pathways are also sensitive to cannabinoid modulation . Norepinephrine release is inhibited by cannabinoid agonists, albeit in a species-specific manner, being reduced in human and guinea pig hippocampus and cortex, but not in rat hippocampus or mouse hippocampus, neocortex, and amygdala . These species differences are intriguing, especially in light of the highly conserved distribution of CB1 receptors on the axon terminals of hippo campal GABAergic and septohippocampal cholinergic neurons . In the rat locus coeruleus, where ascending noradrenergic pathways originate, CB1 mRNA expression is very low . Thus it would be interesting to determine whether a diverging pattern of CB1 receptor expression might explain the greater sensitivity of human and guinea pig noradrenergic transmission to cannabinoid regulation.The finding that cannabinoid agonists reduce both electrically and Ca2-evoked serotonin release in mouse brain cortical slices accords with the prevailing presynaptic localization of CB1 receptors. But whether serotonergic terminals do in fact contain such receptors is still unknown. Notably, the observed maximal reduction in serotonin release is quite low compared with other transmitters like acetylcholine or GABA . Furthermore,marijuana grow system in situ hybridization studies in the raphe nuclei have yielded inconsistent results , and immunohistochemical investigations have not yet been reported.
As we have already pointed out, CB1 receptors are located on the axon terminals of a specific GABAergic cell population in several cortical networks characterized by the expression of CCK . Therefore, it is not unexpected that cannabinoid agonists inhibit potassium-evoked CCK release in rat hippocampal slices . More surprising, however, and still unexplained, is the observation that CCK release is unchanged in the frontal cortex . This discrepancy is surprising in light of the coexpression of CB1 and CCK in the entire neocortex and in the hippocampus . In addition, the observed maximal reduction of cholecystokinin was only 40% in the hippocampus, which seems to be quite low considering the fact that nearly all CCK-containing axon terminals carry CB1 receptors in this brain region . Clarifying this point is particularly important in view of the possible interactions of CCK and anandamide in regulating anxiety and other emotional states .CB1 cannabinoid receptors are expressed by at least three different GABAergic cell populations in the striatum . Hence, cannabinoid effects on GABA release in different regions of the basal ganglia are well documented, and solid evidence for presynaptic cannabinoid receptors on GABAergic axon terminals derives from several different pharmacological approaches. Application of cannabinoid agonists to parasagittal slices of the rat midbrain causes a significant reduction of GABAA receptor-mediated currents recorded in sustantia nigra pars reticulata neurons after stimulation of the internal capsule . Comparable results were obtained in coronal midbrain sections, although in this preparation GABAergic currents are more likely to derive from local GABAergic interneurons . The presynaptic nature of these responses is supported by the increased paired pulse ratio of evoked IPSCs and by the lack of cannabinoid modulation on GABAA receptor-mediated currents elicited by bath application of GABA . In vivo experiments have provided additional insight on the roles of CB1 receptors on striatonigral GABAergic terminals . Both systemically and locally applied cannabinoid agonists increase spontaneous activity of substantia nigra pars reticulata neurons, probably by removing an ongoing GABAergic inhibition .
Moreover, striatal stimulation inhibits the firing of nigral neurons, which is also alleviated by cannabinoids. Blocking of GABAA receptors by bicuculline reverses this effect, indicating that cannabinoid treatment suppresses GABA release . Striatal stimulation also results in reduced firing of pallidal neurons, and this effect is antagonized by systemic administration of a cannabinoid agonist . Surprisingly, local administration of the compound into the globus pallidus does not reverse this effect, raising doubts as to the role of striatopallidal GABAergic projections . Although cannabinoid binding is lower in the striatum compared with its output structures, it is still quite abundant . In addition, endocannabinoid release and local cannabinoid receptors may participate in the modulation of striatal neuronal activity . Szabo´ et al. provided electrophysiological evidence that cannabinoids inhibit the amplitude of IPSCs recorded from medium spiny neurons. One presumptive site for this action is the axon terminals of intrinsic inhibitory interneurons , which provide the major inhibitory control over the activity of striatal projection neurons . The contribution of recurrent axon collaterals of medium spiny neurons cannot be excluded at present. In the shell of the nucleus accumbens, cannabimimetic agents decrease the amplitude of evoked IPSCs and increase the paired-pulse ratio, but do not alter the amplitude of miniature IPSCs, indicating a presynaptic inhibitory effect on GABA release . In situ hybridization and immunostaining studies of this region report low CB1 receptor levels , but this low signal may simply reflect a restricted distribution of the receptor to select interneuronal subtypes, as is the case elsewhere in the CNS. The important functions served by the nucleus accumbens in motivational and reward processes and the impact that cannabinoid drugs exert on such processes should encourage further studies aimed at establishing the precise localization of CB1 receptors in this structure.The glutamatergic innervation of the basal ganglia derives from three main sources. Neurons in the striatum receive glutamatergic axon terminals from cortical and thalamic projection neurons, whereas neurons in the substantia nigra pars reticulata and globus pallidus receive glutamatergic input from the subthalamic nucleus. Both pathways can be modulated by cannabimimetic agents, which inhibit excitatory postsynaptic currents in the striatum as well as the substantia nigra pars reticulata .
The increased paired-pulse ratio and coefficient of variation, together with the lack of effect of cannabinoids on response to bath-applied glutamate, support a presynaptic site of action. In addition, currents evoked by direct glutamate application are not modulated by cannabinoids, demonstrating the lack of a postsynaptic component in these effects. A recent study by Gerdeman et al. provided definitive evidence that the reduction of glutamate release in the striatum is mediated by CB1 receptors,cannabis vertical farming by showing that this effect is absent in CB1 /_x0005_ mice. Furthermore, these authors also demonstrated that the ability of cannabinoid agonists to acutely inhibit glutamate release is a crucial factor in the initiation of striatal long-term depression , a form of synaptic plasticity characterized by a persistent diminution in excitatory transmission. The cannabinoid modulation of glutamatergic neurotransmission in the globus pallidus and the substantia nigra pars reticulata may be of considerable functional importance . Indeed, in contrast to striatal GABAergic projections to the output nuclei, which are usually quiescent, the subthalamic glutamatergic innervation to these two structures is tonically active. The administration of cannabinoid agonists produces changes in the firing of pallidal and nigral neurons, which are consistent with a decrease in this intrinsic activity . It will be interesting to determine whether the endocannabinoid system plays a similar role and, if so, under which physiological circumstances. In the nucleus accumbens, cannabinoid agonists reduce the amplitude of field excitatory postsynaptic potentials as well as EPSCs recorded from medium spiny neurons in the core, but not the shell region of this nucleus . The relatively high cannabinoid concentrations required to produce these effects, and the low expression of CB1 receptors in the projection neurons of the prefrontal cortex, basolateral amygdala, and thalamus that innervate the nucleus accumbens, suggest that in this region, as in the hippocampus , cannabimimetic agents target a CB1-like receptor distinct from CB1. However, the possibility that the reduced cannabinoid sensitivity may reflect the very low expression level of CB1 receptors in glutamatergic neurons cannot be excluded, and recent experiments demonstrating that evoked EPCS are not modulated by cannabinoids in CB1 knock-out mice may also favor this explanation .The cerebellum contains one of the highest densities of CB1 receptors in the brain. Expression of these receptors in local GABAergic interneurons has been suggested by many studies, whereas Purkinje cells do not contain CB1 mRNA . Immunostainings revealed CB1-positive putative GABAergic axon terminals forming a pericellular matrix around the axon initial segment and cell body of Purkinje cells or impinging upon their dendritic tree . In accordance, GABAergic synaptic currents recorded from Purkinje cells are strongly modulated by cannabinoids.
Takahashi and Linden provided the first evidence that spontaneous IPSCs are suppressed by cannabinoid agonists. They estimated that the amplitude of action potential-dependent IPSCs is reduced by 75%, whereas the amplitude of miniature IPSCs is not affected, suggesting a presynaptic mechanism of action. Subsequent experiments using paired recording and imaging of calcium transients in inhibitory axon terminals confirmed this observation and extended it, by showing that endocannabinoids may also regulate afferent inhibitory inputs to Purkinje cells in a retrograde manner . Verifying the role of CB1 receptors, this response is absent from CB1 /_x0005_ mice .In the cerebellum, as in many other brain regions, cannabinoids can effectively modulate neurotransmission not only at inhibitory but also at excitatory synapses. Two pathways provide excitatory input to the cerebellum, the climbing fibers originating from the inferior olive and the parallel fibers deriving from local glutamatergic granule cells. Early anatomical studies reported a high density of cannabinoid binding sites in the molecular layer of the cerebellar cortex along with the expression of CB1 mRNA in granule cells , indicating, but not proving, the presence of CB1 receptors on parallel fibers. Subsequent immunohistochemical experiments supported this notion by revealing a dense CB1-positive axonal mesh work in the molecular layer . Hence, CB1 receptors are situated in a central position to modulate the excitatory input of Purkinje cells. Indeed, whole cell patch-clamp studies revealed that cannabinoids effectively decrease parallel fiber EPSCs . Experimental evidence supports a presynaptic effect, in which activation of cannabinoids results in a reduced probability of glutamate release. Neither the excitability of parallel fibers nor the response to locally applied glutamate was modified by cannabinoid receptor agonists . Moreover, although the frequency of miniature EPSCs was decreased, the amplitude was also unchanged, indicating the presynaptic localization of cannabinoid receptors. An important consequence of this phenomenon is that cannabinoids may impair cerebellar long-term depression . In addition, recent experiments uncovered that endocannabinoids also serve as retrograde signaling molecules in DSE, a phenomenon discussed in detail in section V . In contrast to the cannabinoid effects on parallel fibers, the localization of cannabinoid receptors on climbing fibers seems to be more modest. While a cannabinoidagonist strongly reduced the amplitude of parallel fiber EPSCs , EPSCs deriving from putative climbing fibers were only slightly modulated . However, experimental evidence shows that modulation of glutamate release at climbing fibers is also under the control of endocannabinoids released by the postsynaptic Purkinje cells . In this case, the role of CB1 receptors is not clear yet, because only low levels of CB1 mRNA were found in the inferior olive, where climbing fibers originate . Recent data also suggest the existence of additional cannabinoid binding sites in the cerebellum distinct from CB1, although the molecular identity and precise localization of these putative sites is still unknown .One common feature of the regulatory actions of these compounds is their ability to reduce inhibitory neurotransmission in the rostral ventromedial medulla, the PAG, and the trigeminal nucleus caudatus . Patch-clamp experiments revealed that in these three structures cannabinoid agonists reduce GABA release through a presynaptic mechanism. In the trigeminal nucleus, the release of another inhibitory transmitter, glycine, is also reduced . Although the presynaptic localization of cannabinoid receptors is confirmed by several experiments, the role of CB1 receptors remains equivocal. In addition, detailed studies clarifying the precise localization of CB1 receptors at the subcellular level in these brain areas have not yet been conducted.