We found that (1) levels of CB1R mRNA and protein were significantly reduced and highly correlated in the DLPFC of subjects with schizophrenia; (2) the observed differences in CB1R mRNA expression were significantly correlated with those in GAD67 and CCK mRNA in the same subject pairs, suggesting that downregulation of CB1R in schizophrenia may be a compensatory response to impaired GABA neurotransmission in CCK-containing neurons; and (3) the reductions in CB1R mRNA and protein could not be explained by potential confounding factors.
Several lines of evidence indicate that the reductions in CB1R mRNA and protein levels in schizophrenia are not a consequence of factors frequently associated with the illness. First, CB1R mRNA expression was not altered in the DLPFC of monkeys with long-term exposure to typical or atypical antipsychotics in a manner that mimics the clinical treatment of schizophrenia (). Consistent with these observations, the 4 subjects with schizophrenia who were not receiving antipsychotic medications at the time of death () all had lower CB1R mRNA and protein levels than their matched comparison subjects. In addition, mean CB1R mRNA and protein levels did not differ between the subjects with schizophrenia who were receiving or had stopped receiving antipsychotic medication at the time of death (). Second, neither a diagnosis of substance abuse or dependence nor a history of cannabis use accounted for the group differences in CB1R mRNA or protein levels (). In fact, mean CB1R mRNA and protein levels in those subjects with schizophrenia with a substance use disorder or a history of cannabis use were actually higher than those in subjects who did not meet these criteria, suggesting that these factors might have blunted the decreases in CB1R mRNA and protein levels in schizophrenia. Consistent with these observations, substances of abuse do not affect CB1R mRNA expression or CB1R binding in rodent neocortex (with the exception of cocaine, which only affected CB1R mRNA levels35,36
), and long-term exposure to CB1R agonists either does not alter CB1R mRNA expression37–39
or increases its expression in cortical structures.40
In addition, monkeys with long-term exposure to Δ9-tetrahydrocannabinol or marijuana smoke did not exhibit alterations in CB1R density in the prefrontal cortex.41
However, not all animal studies have produced similar results,42
so an effect of prior cannabis exposure cannot be definitively excluded. Third, the lower CB1R mRNA and protein levels in schizophrenia were not associated with the use of antidepressant medication, benzodiazepines, or valproate at the time of death, death by suicide, or a diagnosis of schizoaffective disorder (). Finally, measures of RNA quality and quantity (RIN and pH) were in the ranges associated with excellent RNA preservation31
in all of the subjects (), and the effect of diagnosis on CB1R measures remained significant when the effects of these variables were controlled statistically. Furthermore, the expression of other transcripts was not altered in these same subjects with schizophrenia,23,26,27
confirming that the observed reductions in CB1R mRNA levels are not attributable to a general loss of mRNA integrity in the subjects with schizophrenia.
In contrast to our findings of reduced CB1R mRNA and protein levels, increased binding of the CB1R agonist [3
H]CP-55940 was reported in the DLPFC4
and posterior cingulate cortex6
of subjects with schizophrenia. Increased [3
H]CP-55940 binding might reflect the presence of an allosteric modulation site on CB1Rs that, when bound, elicits a conformational change in the receptor, increasing the affinity of [3
H]CP-55940 for the orthosteric binding site.43
H]CP-55940 binding can reflect differences other than the amount of receptor present. Increased binding of the CB1R antagonist [3
H]SR141716 was also reported in the anterior cingulate cortex of subjects with schizophrenia.5
Functional effects of SR141716 have been found in CB1R knockout mice, suggesting that it binds receptors other than the CB1R.44,45
Importantly, no studies reporting increased CB1R binding in schizophrenia conducted saturation and competition experiments to determine whether Kd as opposed to Bmax was altered in schizophrenia. Finally, the laminar patterns of radioligand binding in these studies, when reported, are not consistent with those previously reported in human, monkey, or rat neocortex46–48
and do not match the laminar distribution of CB1R-immunoreactive axons in the same regions of monkey and human cortex20
; these findings suggest that the binding of these radioligands does not represent the relative amount of CB1R protein present.
In the neocortex, CB1Rs are heavily localized to inhibitory axon terminals of the subpopulation of GABA basket neurons that contain CCK.19,20
Asymmetric, excitatory, CB1R-immunoreactive synapses have also been observed,49,50
and CB1R agonists modulate glutamate release, consistent with a presynaptic localization of CB1Rs in pyramidal cell axon terminals.51
However, CB1R mRNA levels are much higher in GABA neurons than in pyramidal cells,32
the density of CB1Rs is more than 20-fold higher in inhibitory terminals than in excitatory terminals, and the concentration of CB1R agonist necessary for 50% suppression of glutamate release is approximately 30 times higher than that necessary to suppress GABA release.52
These data indicate that CCK neuron axon terminals contain much higher levels of CB1Rs than pyramidal cell axon terminals and are more sensitive to the effects of CB1R agonists. Importantly, the antibody used in this study exclusively labels symmetric, inhibitory synapses by electron microscopy,20
probably because the level of CB1Rs in excitatory terminals is below the threshold of detectability. Thus, the observed reductions in CB1R protein levels in subjects with schizophrenia are likely to reflect lower CB1R levels specifically in inhibitory neurons and axon terminals rather than in pyramidal neurons and axons. Furthermore, the strong correlations between the differences in CB1R mRNA and GAD67
and CCK mRNA support the interpretation that the observed deficits in CB1R levels reflect changes in GABA neurons and not pyramidal cells.
Disturbances in inhibitory neurotransmission appear to play a prominent role in the dysfunction of the DLPFC of subjects with schizophrenia18
as demonstrated by the consistent finding of an approximately 25% to 35% reduction in the expression of GAD67
mRNA across layers 2 through 5.22,53–55
Parvalbumin-containing interneurons appear to account for the decreased GAD67
mRNA expression in layers 3 and 4,26
and the results of the present study suggest that CB1R- and CCK-containing neurons might contribute to the GAD67
mRNA deficit in layers 2 through superficial 3. In the primate DLPFC, the highest densities of both CB1R- and CCK-positive neurons are found in these layers, and both CB1R- and CCK-positive axon terminals densely innervate layer 4.20,56
In addition, these 2 proteins are colocalized in terminals that furnish perisomatic inputs to pyramidal neurons.19,32,57
Thus, our findings of reduced CB1R mRNA in layers 2 through superficial 3, reduced CB1R immunoreactivity in layer 4, and correlated changes in CB1R, CCK, and GAD67
mRNA in schizophrenia converge on the interpretation that GABA neurotransmission is altered in the subset of CB1R- and CCK-containing GABA neurons that project from the superficial to middle cortical layers.
How might these disturbances be related to the working memory impairments associated with DLPFC dysfunction in schizophrenia? In the human DLPFC, the power of gamma band oscillations (30–80 Hz) increases directly with working memory load,58
and impaired working memory performance in individuals with schizophrenia is associated with reduced frontal lobe gamma band power.59
Neurotransmission of GABA in the DLPFC is essential for both working memory performance16,17
and oscillatory activity.60
Consistent with the anatomical localization of CB1Rs to CCK-containing neuron axon terminals, activation of CB1Rs inhibits GABA release from these terminals and strongly suppresses GABAA
receptor–mediated inhibitory postsynaptic currents in pyramidal neurons.19,57,61
Indeed, the acute activation of CB1Rs with exogenous cannabinoids decreases the power of gamma oscillations in the rodent hippocampus, entorhinal cortex, and prefrontal cortex, presumably by disrupting the synchronous firing of pyramidal neurons.62–64
Thus, the disruption of gamma oscillations by CB1R activation in the DLPFC may explain the impairments in working memory performance in both humans and animals following systemic administration of cannabinoids65–67
; however, this interpretation remains to be tested in primates.
Our findings might represent a downregulation of CB1Rs in response to elevated levels of endocannabinoids in schizophrenia (eg, increased cerebrospinal fluid and blood levels of anandamide1–3
). However, whether the DLPFC contributes to reported elevated levels of anandamide is unknown. Furthermore, 2-arachidonoyl-glycerol, not anandamide, appears to be the principal endocannabinoid for CB1Rs in cortical and hippocampal GABA neurons.68–70
Alternatively, we suggest that the downregulation of CB1R mRNA and protein in schizophrenia may represent a compensatory response to a deficit of GABA synthesis in CCK-containing neurons. That is, a lower density of CB1Rs could, by reducing the endocannabinoid-mediated suppression of GABA release from the perisomatic terminals of CB1R- and CCK-containing interneurons, contribute to a partial normalization of gamma band power and working memory function. Downregulation of CCK expression may also represent a compensatory response that, by reducing CCKB
receptor–mediated enhancement of 2-arachidonoyl-glycerol synthesis, decreases endocannabinoid-mediated suppression of GABA release from CB1R- and CCK-containing axon terminals.71
This interpretation implies that cannabis use in vulnerable individuals would counteract these compensatory responses, providing a potential mechanism linking cannabis exposure with an increased risk for the cognitive impairments of schizophrenia.
This interpretation also suggests possible novel molecular targets for treating the cognitive deficits in schizophrenia. For instance, CB1R antagonists would be predicted to augment the intrinsic compensatory downregulation of CB1R expression, further limit the endocannabinoid-mediated suppression of GABA release from CB1R- and CCK-containing terminals, and enhance the ability of CCK basket neurons to synchronize pyramidal neurons in gamma oscillations. In addition, at least in the hippocampus, GABAA
receptors containing the α2 subunit are selectively located on pyramidal cell bodies postsynaptic to CB1R- and CCK-containing terminals.72
Thus, positive allosteric modulators of the benzodiazepine binding site with selectivity for GABAA
receptors containing the α2 subunit would be predicted to increase the efficacy of GABA released from CB1R- and CCK-containing terminals and might be synergistic with the proposed effects of such agents at augmenting the input from parvalbumin-containing chandelier neurons to the axon initial segment of pyramidal neurons.73,74
Together, such agents might enhance the synchronization of pyramidal neuron activity by restoring normal levels of perisomatic GABA input to pyramidal neurons.