In many neurons more than one peptide is colocalized with a classical neurotransmitter. The functional consequence of such an arrangement has been rarely investigated. Here, within the feeding circuit of Aplysia, we investigate at a single synapse the actions of two modulatory neuropeptides that are present in a cholinergic interneuron. In combination with previous work, our study shows that the command-like neuron for feeding, CBI-2, contains two neuropeptides, feeding circuit activating peptide (FCAP) and cerebral peptide 2 (CP2). Previous studies showed that high-frequency prestimulation or repeated stimulation of CBI-2 increases the size of CBI-2 to B61/62 excitatory postsynaptic potentials (EPSPs) and shortens the latency of firing of neuron B61/62 in response to CBI-2 stimulation. We find that both FCAP and CP2 mimic these two effects. The variance method of quantal analysis indicates that FCAP increases the calculated quantal size (q) and CP2 increases the calculated quantal content (m) of EPSPs. Since the PSP amplitude represents the product of q and m, the joint action of the two peptides is expected to be cooperative. This observation suggests a possible functional implication for multiple neuropeptides colocalized with a classical neurotransmitter in one neuron.

Nitric oxide (NO) acts as a neurotransmitter and neuromodulator in the nervous systems of many vertebrates and invertebrates. We investigated the mechanism of NO action at an identified synapse between a mechanoafferent neuron, C2, and the serotonergic metacerebral cell (MCC) in the cerebral ganglion of the mollusc Aplysia californica. Stimulation of C2 produces a decreasing conductance, very slow EPSP in the MCC. C2 is thought to use histamine and NO as cotransmitters at this synapse, because both agents mimic the membrane responses. Now we provide evidence that treatment with NO donors stimulates soluble guanylyl cyclase (sGC) in the MCC, and as a result cGMP increases. S-Nitrosocysteine (SNC, an NO donor) and 8-bromo-cGMP (8-Br-cGMP) both induced the membrane depolarization and increase in input resistance that are characteristic of the very slow EPSP. Two inhibitors of sGC, 6-anilino-5,8-quinolinequinone (LY83583) and 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxaline-1-one (ODQ), suppressed both the very slow EPSP and the membrane responses to SNC but not the histamine membrane responses. NO-induced cGMP production was determined in the MCC using cGMP immunocytochemistry (cGMP-IR). In the presence of 3-isobutyl-1-methylxanthine (IBMX), 10 microM SNC was sufficient to induce cGMP-IR, and the staining intensity increased as the SNC dose was increased. This cGMP-IR was suppressed by ODQ in a dose-dependent manner and completely blocked by 10 microM ODQ. Histamine did not induce cGMP-IR. The results suggest that NO stimulates sGC-dependent cGMP synthesis in the MCC and that cGMP mediates the membrane responses. The cotransmitter histamine induces essentially the same membrane responses but seems to use a separate and distinct second messenger pathway.

Abnormal phospholipid metabolism has been implicated in the pathogenesis of schizophrenia, and it was reported that phospholipase C (PLC) b1 is reduced in specific brain areas of patients with schizophrenia. However, the causal relationship of the PLCb1 gene with behavioral symptoms of schizophrenia remains unclear. To address this issue, we have examined the mutant mice lacking PLCb1 for schizophrenia-related phenotypes by performing various behavioral tests, including general locomotor activity, sensorimotor gating, social behaviors, and learning and memory. Phospholipase C b1 knockout mice showed hyperactivities in an open field. They showed impaired prepulse inhibition of acoustic startle response, which was ameliorated by a systemic administration of an antipsychotic D2-receptor antagonist, haloperidol. In addition, they showed abnormal social behaviors, such as lack of barbering behavior, socially recessive trait and lack of nesting behavior. Furthermore, they showed impaired performance in the delayed-nonmatch-to-sample T-maze test. The present results show that the PLCb1 mutant mice share some of the behavioral abnormalities that have been reported in patients with schizophrenia. Thus, the PLCb1-linked signaling pathways may be involved in the neural system whose function is disrupted in the pathogenesis of schizophrenia.

Hallucinations and delusions are the most prominent symptoms of schizophrenia and characterized by impaired reality testing. Representation-mediated taste aversion (RMTA) has been proposed as a potential behavioral assessment of reality testing and has been applied to a neurodevelopmental rat model of schizophrenia. However, the theory underlying this approach has not been generalized yet with any demonstration of impaired reality testing in other animal models of schizophrenia, such as genetically-modified mice. We devised a RMTA procedure for mice that combines a Pavlovian association protocol pairing odor conditioned stimulus (CS) with sugar reward unconditioned stimulus (US), and a conditioned taste aversion (CTA) method. In this RMTA paradigm, we compared performances of wild-type (PLCβ1+/+) mice and phospholipase C β1 knock-out (PLCβ1-/-) mice which are known as one of the genetic models for schizophrenia. With a minimal amount of initial odor-sugar associative training, both PLCβ1+/+ and PLCβ1-/- mice were able to form an aversion to the sugar reward when the odor CS predicting sugar was paired with nausea. With an extended initial training, however, only PLCβ1-/- mice could form a RMTA. This persistent RMTA displayed by PLCβ1-/- mice shows their inability to distinguish real sugar from the CS-evoked representation of sugar at a stage in associative learning where wild-type mice normally could differentiate the two. These results demonstrate an impaired reality testing first observed in a genetic mouse model of schizophrenia, and suggest that RMTA paradigm may, with general applicability, allow diverse biological approaches to impaired reality testing.

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Vascularization of tissues, organoids and organ-on-chip models has been attempted using endothelial cells. However, the cultured endothelial cells lack the capacity to interact with other somatic cell types, which is distinct from developing vascular cells in vivo. Recently, it was demonstrated that blood vessel organoids (BVOs) recreate the structure and functions of developing human blood vessels. However, the tissue-specific adaptability of BVOs had not been assessed in somatic tissues. Herein, we investigated whether BVOs infiltrate human cerebral organoids and form a blood–brain barrier. As a result, vascular cells arising from BVOs penetrated the cerebral organoids and developed a vessel-like architecture composed of CD31+ endothelial tubes coated with SMA+ or PDGFR+ mural cells. Molecular markers of the blood-brain barrier were detected in the vascularized cerebral organoids. We revealed that BVOs can form neural-specific blood-vessel networks that can be maintained for over 50 days.

Koh H-Y, Weiss KR. Activity-dependent peptidergic modulation of the plateau-generating neuron B64 in the feeding network of Aplysia. J Neurophysiol 97: 1862-1867, 2007. First published January 3, 2007 doi:10.1152/jn.01230.2006. Many behaviors display various forms of activity-dependent plasticity. An example of such plasticity is the progressive shortening of the duration of protraction phase of feeding responses of Aplysia that occurs when feeding responses are repeatedly elicited. A similar protraction-duration shortening is observed in isolated ganglia of Aplysia when feeding-like motor programs are elicited through a prolonged stimulation of the command-like neuron CBI-2. Here, we investigate a cellular mechanism that may underlie this activity-dependent shortening of protraction duration of feeding motor programs. CBI-2 contains two neuropeptides, CP2 and FCAP. Previous work showed that CP2 shortens protraction duration of CBI-2 elicited programs. We show here that the same is true for FCAP. We also show that both CP2 and FCAP modulated the biophysical properties of a plateaugenerating neuron, B64, that plays an important role in terminating the protraction phase of feeding motor programs. We find that prestimulation of CBI-2, as well as superfusion of CP2 and FCAP, lowered the threshold for activation of the plateau potential in B64. The threshold-lowering actions of CBI-2 prestimulation were occluded by superfusion of FCAP and CP2. Furthermore, at elevated temperature, conditions under which peptide release is prevented in Aplysia, prestimulation of CBI-2 does not lower the plateaupotential threshold, whereas superfusion of CP2 and FCAP does. Our findings are consistent with the hypothesis that peptides released from CBI-2 lower the threshold for activation of plateau potential in B64, thereby contributing to the shortening of protraction duration when CBI-2 is repeatedly activated.

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