Reversible DREADD inactivation of orbitofrontal cortex neurons in rhesus monkeys with contralateral rhinal cortex removal disrupts cued reward discrimination. II. Histological analysis.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are G-protein coupled receptors specifically activated by clozapine-N-oxide (CNO), an otherwise inert ligand. In the mammalian brain neurons expressing DREADDs can be inhibited or excited (dependent on the DREADD) by systemic delivery of CNO, no matter where they are located. The hM4Di receptor is a DREADD that inhibits neuronal firing via a G-protein coupled potassium channel on binding of CNO. We created a lentivirus-construct with a human synapsin promoter driving expression of the hM4Di receptor. Using a handheld syringe we injected the resulting lentivirus into monkey orbitofrontal cortex (OFC) of one hemisphere in each of two monkeys that already had a contralateral rhinal cortex (Rh) removal. As shown in the accompanying poster, activation of the DREADD with parenterally delivered CNO did not have any effect on monkeys with unilateral Rh lesions, whereas there was a significant change in behavior in the two DREADD expressing monkeys on the day of activation. We used antibody staining against the DREADD expressed by the lentivirus to identify the areal coverage with cellular expression of hM4Di on a flattened representation of OFC. Expression in any layer was projected onto a line running through layer 4. Our approximately 60 punctate injections led to expression within at least some layers to cover about 10% of the OFC. We did not observe any cortical layer preference for expressing neurons. The expression widths for individual injections varied from 0.3 - 3.0 mm. Preliminary analysis using confocal microscopy shows co-expression of the DREADD with the neuronal maker NeuN in greater than 80% of neurons in areas between 0.2 - 2 mm surrounding the injection tracks (high-density expression areas). Outside the high-density areas neuronal somatic expression fell sharply. For many injection sites a bloom of fibers was visible for several millimeters around the somatic expression areas. We tested the functionality of our lentivirus construct by transducing primary neuron cultures. Introducing CNO into the culture bath significantly decreased neuronal spontaneous discharge. Washout of the CNO with normal culture medium was followed by a restoration of neuronal activity. In our Rh-OFC disconnection design, i.e., unilateral rhinal removal with contralateral OFC DREADD neuronal silencing, it appears that inhibition of fewer than 10% of neurons, spaced in an irregular lattice throughout the OFC, is sufficient to disrupt cued reward discrimination behavior in monkeys reversibly.Society for Neuroscienc