Opioid receptors are known to undergo complex regulatory changes in response to ligand exposure. In the present study, we examined the effect of morphine on the in vitro and in vivo density and trafficking of delta opioid receptors (deltaORs). Prolonged exposure (48 hr) of cortical neurons in culture to morphine (10 microm) resulted in a robust increase in the internalization of Fluo-deltorphin, a highly selective fluorescent deltaOR agonist. This effect was mu-mediated because it was entirely blocked by the selective mu opioid receptor antagonist d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH(2) and was reproduced using the selective mu agonist fentanyl citrate. Immunogold electron microscopy revealed a marked increase in the cell surface density of deltaORs in neurons exposed to morphine, indicating that the increase in Fluo-deltorphin internalization was caused by increased receptor availability. Prolonged morphine exposure had no effect on deltaOR protein levels, as assessed by immunocytochemistry and Western blotting, suggesting that the increase in bioavailable deltaORs was caused by recruitment of reserve receptors from intracellular stores and not from receptor neosynthesis. Complementary in vivo studies demonstrated that chronic treatment of adult rats with morphine (5-15 mg/kg, s.c., every 12 hr) similarly augmented targeting of deltaORs to neuronal plasma membranes in the dorsal horn of the spinal cord. Furthermore, this treatment markedly potentiated intrathecal d-[Ala(2)]deltorphin II-induced antinociception. Taken together, these results demonstrate that prolonged stimulation of neurons with morphine markedly increases recruitment of intracellular deltaORs to the cell surface, both in vitro and in vivo. We propose that this type of receptor subtype cross-mobilization may widen the transduction repertoire of G-protein-coupled receptors and offer new therapeutic strategies.

The binding, internalization, and trafficking of the fluorescently labeled opioid peptides Fluo-dermorphin and Fluo-deltorphin were quantitatively studied by confocal microscopy in primary cortical neurons in culture. Specific binding of these selective ligands to neurons naturally expressing mu (mu) and delta (delta) opioid receptors (OR), respectively, resulted in their internalization into neuronal somas and processes, as indicated by the persistence of fluorescent labeling following removal of cell surface binding by hypertonic acid wash. This internalization was receptor-specific, as the fluorescent signal was completely abolished when the cells were concomitantly incubated with the opioid receptor antagonist naloxone. It also was clathrin-dependent, as it was totally prevented by the endocytosis inhibitor phenylarsine oxide. Accordingly, internalized ligands were detected inside small, endosome-like vesicles. These labeled vesicles accumulated within nerve cell bodies between 5-30 min of incubation with the fluorescent ligands. This accumulation was abolished after treatment with the antitubular agent nocodazole, suggesting that it was due to a microtubule-dependent, retrograde transport of the internalized ligands from processes to the soma. By contrast, there was no change in the compartmentalization of internalized (mu)OR or deltaOR, as assessed by immunocytochemistry, suggesting that the latter were recycled locally. The present results provide the first demonstration of receptor-mediated internalization of opioid peptides in cultured neurons. It is proposed that their retrograde transport into target cells might be involved in mediating some of the long-term, transcriptional effects of opioids.

An inductorless MedRadio (413∼419 MHz) OOK transceiver for an implantable brain-machine interface (BMI) in 180 nm CMOS is presented. An event-driven supply modulation (EDSM) technique is introduced in the receiver to lower the power consumption to 42/92 μW at 1/10 kbps, achieving -79/-74 dBm sensitivity (at 0.1% BER). The transmitter employs a current-starved ring oscillator with an automatic frequency calibration loop, achieving 14% efficiency for random OOK data at -4 dBm output power. Wireless connection, benchtop, multi-user coexistence, and in-vitro phantom test results are demonstrated.

A MedRadio 413-419-MHz inductorless transceiver (TRX) for an implantable brain-machine interface (BMI) in a 180 nm-CMOS process is presented. Occupying 5.29 mm2 of die area (including pad ring), this on-off keying (OOK) TRX employs a non-coherent direct-detection receiver (RX), which exhibits a measured in-band noise figure (NF) of 4.9 dB and S11 of-13.5 dB. An event-driven supply modulation (EDSM) technique is introduced to dramatically lower the RX power consumption. Incorporating an adaptive feedback loop, this RX consumes 42-/92-μW power from 1.8-V supply at 1/10-kbps data rates, achieving-79/-74-dBm sensitivities for 0.1% bit error rate (BER). The TX employs a current starved ring oscillator with an automatic frequency calibration loop, covering 9% supply voltage variation and 15 °C-78 °C temperature range that guarantees operation within the emission mask. The direct-modulation TX achieves 14% efficiency for a random OOK data sequence at-4-dBm output power. Wireless testing over a 350-cm distance accounting for bio-signal data transfer, multi-user coexistence, and in vitro phantom measurement results is demonstrated.

Two brain signal acquisition (BSA) front-ends incorporating two CMOS ultralow power, low-noise amplifier arrays and serializers operating in mosfet weak inversion region are presented. To boost the amplifier's gain for a given current budget, cross-coupled-pair active load topology is used in the first stages of these two amplifiers. These two BSA front-ends are fabricated in 130 and 180 nm CMOS processes, occupying 5.45 mm ² and 0.352 mm ² of die areas, respectively (excluding pad rings). The CMOS 130-nm amplifier array is comprised of 64 elements, where each amplifier element consumes 0.216 μW from 0.4 V supply, has input-referred noise voltage (IRNoise) of 2.19 μV[Formula: see text] corresponding to a power efficiency factor (PEF) of 11.7, and occupies 0.044 mm ² of die area. The CMOS 180 nm amplifier array employs 4 elements, where each element consumes 0.69 μW from 0.6 V supply with IRNoise of 2.3 μV[Formula: see text] (corresponding to a PEF of 31.3) and 0.051 mm ² of die area. Noninvasive electroencephalographic and invasive electrocorticographic signals were recorded real time directly on able-bodied human subjects, showing feasibility of using these analog front-ends for future fully implantable BSA and brain- computer interface systems.