Isolated Mouse Dorsal Root Ganglion Research Articles

Analgesic Buxus alkaloids with Enhanced Selectivity for the Low-Voltage-Gated Calcium Channel Cav 3.2 over Cav 3.1 through a New Binding Mode.

Low-voltage-gated calcium channels (LVGCCs; Cav 3.1-3.3) represent promising drug targets for epilepsy, pain, and essential tremor. At present, modulators with heightened selectivity for a subtype of LVGCCs are still highly desired. In this study we explored three classes of Buxus alkaloids and identified 9(10/19)abeo-artanes Buxusemine H and Buxusemine L (BXSL) as an unprecedented type of Cav 3.2 inhibitors. Particularly, BXSL exhibited Cav 3.2 inhibition comparable to Z944, a non-subtype-selective LVGCCs inhibitor under clinical trial. While lacking specificity for Cav 3.3, BXSL showed a 30-fold selectivity of Cav 3.2 over Cav 3.1. As compared to several well-known inhibitors, the experimental and computational studies suggested BXSL exhibits a distinct binding mode to Cav 3.2, notably through the essential interaction with serine-1543 in domain III. Furthermore, BXSL showed minimal impact on various recombinant and native nociceptive ion channels, while significantly reducing the excitability of isolated mouse dorsal root ganglion neurons. Animal studies in wild-type and Cav 3.2 knock-out mice revealed that BXSL (5 mg/kg), by inhibiting Cav 3.2, exhibits an analgesic effect equivalent to Z944 (10 mg/kg) or mibefradil (10 mg/kg). Moreover, we proposed a structural rationale for the high selectivity of 9(10/19)abeo-artane-type alkaloids towards Cav 3.2 over Cav 3.1. This study introduces a novel analgesic agent and valuable molecular insight for structure-based innovative Cav 3.2 drug development.

(-)-Naringenin 4',7-dimethyl Ether Isolated from Nardostachys jatamansi Relieves Pain through Inhibition of Multiple Channels.

(−)-Naringenin 4′,7-dimethyl ether ((−)-NRG-DM) was isolated for the first time by our lab from Nardostachys jatamansi DC, a traditional medicinal plant frequently used to attenuate pain in Asia. As a natural derivative of analgesic, the current study was designed to test the potential analgesic activity of (−)-NRG-DM and its implicated mechanism. The analgesic activity of (−)-NRG-DM was assessed in a formalin-induced mouse inflammatory pain model and mustard oil-induced mouse colorectal pain model, in which the mice were intraperitoneally administrated with vehicle or (−)-NRG-DM (30 or 50 mg/kg) (n = 10 for each group). Our data showed that (−)-NRG-DM can dose dependently (30~50 mg/kg) relieve the pain behaviors. Notably, (−)-NRG-DM did not affect motor coordination in mice evaluated by the rotarod test, in which the animals were intraperitoneally injected with vehicle or (−)-NRG-DM (100, 200, or 400 mg/kg) (n = 10 for each group). In acutely isolated mouse dorsal root ganglion neurons, (−)-NRG-DM (1~30 μM) potently dampened the stimulated firing, reduced the action potential threshold and amplitude. In addition, the neuronal delayed rectifier potassium currents (IK) and voltage-gated sodium currents (INa) were significantly suppressed. Consistently, (−)-NRG-DM dramatically inhibited heterologously expressed Kv2.1 and Nav1.8 channels which represent the major components of the endogenous IK and INa. A pharmacokinetic study revealed the plasma concentration of (−)-NRG-DM is around 7 µM, which was higher than the effective concentrations for the IK and INa. Taken together, our study showed that (−)-NRG-DM is a potential analgesic candidate with inhibition of multiple neuronal channels (mediating IK and INa).

Magnolol attenuates inflammatory pain by inhibiting sodium currents in mouse dorsal root ganglion neurons.

Voltage-gated sodium channels are currently recognized as one of the targets of analgesics. Magnolol (Mag), an active component isolated from Magnolia officinalis, has been reported to exhibit analgesic effects. The objective of this study was to investigate whether the analgesic effect of Mag was associated with blocking Na+ channels. Inflammatory pain was induced by the injection of carrageenan into the hind paw of mice. Mag was administered orally. Mechanical hyperanalgesia was evaluated by using von Frey filaments. Na+ currents and neuronal excitability in acutely isolated mouse dorsal root ganglion (DRG) neurons were recorded with the whole-cell patch clamp technique. Results showed that Mag (10~40mg/kg) dose-dependently inhibited the paw edema and reduced mechanical pain in the inflammatory animal model. Injection of carrageenan significantly increased the amplitudes of TTX-sensitive and TTX-resistant Na+ currents. Compared with the carrageenan group, Mag inhibited the upregulation of two types of Na+ currents induced by carrageenan in a dose-dependent manner. Mag 40mg/kg shifted the inactivation curves of two types of Na+ currents to hyperpolarization and returned to normal animal level without changing their activation curves. Mag 40mg/kg significantly reduced the percentage of cells firing multiple spikes and inhibited the neuronal hyperexcitability induced by carrageenan. Our data suggest that the analgesic effect of Mag may be associated with a decreased neuronal excitability by blocking Na+ current.

Magnolol inhibits sodium currents in freshly isolated mouse dorsal root ganglion neurons.

The voltage-gated sodium channel (VGSC) currents in dorsal root ganglion (DRG) neurons contain mainly TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na+ currents. Magnolol (Mag), a hydroxylated biphenyl compound isolated from the bark of Magnolia officinalis, has been well documented to exhibit analgesic effects, but its mechanism is not yet fully understood. The aim of the present study was to investigate whether the antinociceptive effects of Mag is through inhibition of Na+ currents. Na+ currents in freshly isolated mouse DRG neurons were recorded with the whole cell patch clamp technique. Results showed that Mag inhibited TTX-S and TTX-R Na+ currents in a concentration-dependent manner. The IC50 values for block of TTX-S and TTX-R Na+ currents were 9.4 and 7.0μmol/L, respectively. Therefore, TTX-R Na+ current was more susceptible to Mag than TTX-S Na+ current. For TTX-S Na+ channel, 10μmol/L Mag shifted the steady state inactivation curve toward more negative by 9.8mV, without affecting the activation curve. For TTX-R Na+ channel, 7μmol/L Mag shifted the steady state activation and inactivation curves toward more positive and negative potentials by 6.5 and 11.7mV, respectively. In addition, Mag significantly postponed recovery of TTX-S and TTX-R Na+ currents from inactivation, and produced frequency dependent blocks of both subtypes of Na+ currents. These results suggest that the inhibitory effects of Mag on Na+ channels may contribute to its analgesic effect.

The Distributions of Voltage-Gated K+ current Subtypes in Different Cell Sizes from Adult Mouse Dorsal Root Ganglia.

Voltage-gated K+ (KV) currents play a crucial role in regulating pain by controlling neuronal excitability, and are divided into transient A-type currents (IA) and delayed rectifier currents (IK). The dorsal root ganglion (DRG) neurons are heterogeneous and the subtypes of KV currents display different levels in distinct cell sizes. To observe correlations of the subtypes of KV currents with DRG cell sizes, KV currents were recorded by whole-cell patch clamp in freshly isolated mouse DRG neurons. Results showed that IA occupied a high proportion in KV currents in medium- and large-diameter DRG neurons, whereas IK possessed a larger proportion of KV currents in small-diameter DRG neurons. A lower correlation was found between the proportion of IA or IK in KV currents and cell sizes. These data suggest that IA channels are mainly expressed in medium and large cells and IK channels are predominantly expressed in small cells.

Inhibitory Effects of Honokiol on the Voltage-Gated Potassium Channels in Freshly Isolated Mouse Dorsal Root Ganglion Neurons.

Voltage-gated potassium (KV) currents, subdivided into rapidly inactivating A-type currents (I A) and slowly inactivating delayed rectifier currents (I K), play a fundamental role in modulating pain by controlling neuronal excitability. The effects of Honokiol (Hon), a natural biphenolic compound derived from Magnolia officinalis, on KV currents were investigated in freshly isolated mouse dorsal root ganglion neurons using the whole-cell patch clamp technique. Results showed that Hon inhibited I A and I K in concentration-dependent manner. The IC50 values for block of I A and I K were 30.5 and 25.7µM, respectively. Hon (30µM) shifted the steady-state activation curves of I A and I K to positive potentials by 17.6 and 16.7mV, whereas inactivation and recovery from the inactivated state of I A were unaffected. These results suggest that Hon preferentially interacts with the active states of the I A and I K channels, and has no effect on the resting state and inactivated state of the I A channel. Blockade on K+ channels by Hon may contribute to its antinociceptive effect, especially anti-inflammatory pain.

TRPM8 is a neuronal osmosensor that regulates eye blinking in mice.

Specific peripheral sensory neurons respond to increases in extracellular osmolality but the mechanism responsible for excitation is unknown. Here we show that small increases in osmolality excite isolated mouse dorsal root ganglion (DRG) and trigeminal ganglion (TG) neurons expressing the cold-sensitive TRPM8 channel (transient receptor potential channel, subfamily M, member 8). Hyperosmotic responses were abolished by TRPM8 antagonists, and were absent in DRG and TG neurons isolated from Trpm8−/− mice. Heterologously expressed TRPM8 was activated by increased osmolality around physiological levels and inhibited by reduced osmolality. Electrophysiological studies in a mouse corneal preparation demonstrated that osmolality regulated the electrical activity of TRPM8-expressing corneal afferent neurons. Finally, the frequency of eye blinks was reduced in Trpm8−/− compared with wild-type mice and topical administration of a TRPM8 antagonist reduced blinking in wild-type mice. Our findings identify TRPM8 as a peripheral osmosensor responsible for the regulation of normal eye-blinking in mice.

H2S and Pain: A Novel Aspect for Processing of Somatic, Visceral and Neuropathic Pain Signals.

Hydrogen sulfide (H2S) formed by multiple enzymes including cystathionine-γ-lyase (CSE) targets Cav3.2 T-type Ca2+ channels (T-channels) and transient receptor potential ankyrin-1 (TRPA1). Intraplantar and intracolonic administration of H2S donors promotes somatic and visceral pain, respectively, via activation of Cav3.2 and TRPA1 in rats and/or mice. Injection of H2S donors into the plantar tissues, pancreatic duct, colonic lumen, or bladder causes T-channel-dependent excitation of nociceptors, determined as phosphorylation of ERK or expression of Fos in the spinal dorsal horn. Electrophysiological studies demonstrate that exogenous and/or endogenous H2S facilitates membrane currents through T-channels in NG108-15 cells and isolated mouse dorsal root ganglion (DRG) neurons that abundantly express Cav3.2 and also in Cav3.2-transfected HEK293 cells. In mice with cerulein-induced pancreatitis and cyclophosphamide-induced cystitis, visceral pain and/or referred hyperalgesia are inhibited by CSE inhibitors and by pharmacological blockade or genetic silencing of Cav3.2, and CSE protein is upregulated in the pancreas and bladder. In rats with neuropathy induced by L5 spinal nerve cutting or by repeated administration of paclitaxel, an anticancer drug, the neuropathic hyperalgesia is reversed by inhibitors of CSE or T-channels and by silencing of Cav3.2. Upregulation of Cav3.2 protein in DRG is detectable in the former, but not in the latter, neuropathic pain models. Thus, H2S appears to function as a nociceptive messenger by facilitating functions of Cav3.2 and TRPA1, and the enhanced function of the CSE/H2S/Cav3.2 pathway is considered to be involved in the pancreatitis- and cystitis-related pain and in neuropathic pain.

Sensitization of cutaneous neuronal purinergic receptors contributes to endothelin-1-induced mechanical hypersensitivity

Endothelin (ET-1), an endogenous peptide with a prominent role in cutaneous pain, causes mechanical hypersensitivity in the rat hind paw, partly through mechanisms involving local release of algogenic molecules in the skin. The present study investigated involvement of cutaneous ATP, which contributes to pain in numerous animal models. Pre-exposure of ND7/104 immortalized sensory neurons to ET-1 (30nM) for 10min increased the proportion of cells responding to ATP (2μM) with an increase in intracellular calcium, an effect prevented by the ETA receptor–selective antagonist BQ-123. ET-1 (3nM) pre-exposure also increased the proportion of isolated mouse dorsal root ganglion neurons responding to ATP (0.2–0.4μM). Blocking ET-1-evoked increases in intracellular calcium with the IP3 receptor antagonist 2-APB did not inhibit sensitization to ATP, indicating a mechanism independent of ET-1-mediated intracellular calcium increases. ET-1-sensitized ATP calcium responses were largely abolished in the absence of extracellular calcium, implicating ionotropic P2X receptors. Experiments using quantitative polymerase chain reaction and receptor-selective ligands in ND7/104 showed that ET-1-induced sensitization most likely involves the P2X4 receptor subtype. ET-1-sensitized calcium responses to ATP were strongly inhibited by broad-spectrum (TNP-ATP) and P2X4-selective (5-BDBD) antagonists, but not antagonists for other P2X subtypes. TNP-ATP and 5-BDBD also significantly inhibited ET-1-induced mechanical sensitization in the rat hind paw, supporting a role for purinergic receptor sensitization in vivo. These data provide evidence that mechanical hypersensitivity caused by cutaneous ET-1 involves an increase in the neuronal sensitivity to ATP in the skin, possibly due to sensitization of P2X4 receptors.

Effects of estradiol on voltage-gated sodium channels in mouse dorsal root ganglion neurons

Estrogen has multiple actions in the brain to modulate homeostasis, synaptic plasticity, neuroprotection and pain sensitivity. Previous studies have demonstrated that estradiol may affect the ion channel function. The role of voltage-gated sodium channels in the transmission of nociceptive and neuropathic pain messages is well-established. Herein, we report the effects of estradiol (E2) on TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na+ currents, using a conventional whole-cell patch clamp technique from acutely isolated mouse dorsal root ganglion neurons. We found that the extracellularly 17β-E2 inhibited TTX-S Na+ currents and TTX-R Na+ currents; the effects were rapid, reversible and in a concentration-dependent manner. Moreover, 17β-E2 did not significantly affect the activation curve for Na+ channel, and shifted the steady-state inactivation curve for TTX-S and TTX-R Na+ channels in the hyperpolarizing direction. We also found that the membrane impermeable E2-BSA was as efficacious as 17β-E2, whereas 17α-E2 had no effect. Blockers of PKC (GÖ-6983) and PKA (H-89) abrogated these acute effects of 17β-E2. In conclusion, E2 inhibited voltage-gated Na+ channels in mouse DRG neurons through a membrane ER-activated PKC–PKA signaling pathway. Through the modulation of voltage-gated sodium currents, estradiol could affect cell excitability, firing properties.

Inhibition of voltage-gated sodium channels by bisphenol A in mouse dorsal root ganglion neurons

Bisphenol A (BPA), an estrogenic compound, is contained in cans, polycarbonate bottles, and some dental sealants. Exposure to BPA might have potential toxicological effects on the nervous system. Previous studies have demonstrated that BPA may affect ion channel function, but the effects of BPA on voltage-gated sodium channels are unknown. Herein, we report the effects of BPA on TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na + currents, using a conventional whole-cell patch clamp technique from acutely isolated mouse dorsal root ganglion neurons. BPA inhibited TTX-S Na + currents and TTX-R Na + currents, the effects of BPA were rapid, reversible and in a concentration-dependent manner. Moreover, BPA could shift the voltage-gated activation curve for TTX-S Na + channel in the hyperpolarizing direction without changing that for TTX-R Na + channel; shift the steady-state inactivation curve for TTX-S Na + channel in the depolarizing direction without changing that for TTX-R Na + channel; and lengthen the time course of recovery from inactivation for both TTX-S Na + current and TTX-R Na + current. We also found that PKC inhibitor GÖ-6983 and PKA inhibitor H-89 blocked the BPA-induced inhibition of Na + currents. Considering its complex modulatory effects on voltage-gated sodium channels, BPA might have potential toxicological effects on the nervous system and lead to a change in excitability of nociceptive afferent fibers.

Histamine potentiates acid-induced responses mediating transient receptor potential V1 in mouse primary sensory neurons

In inflamed tissues, extracellular pH decreases and acidosis is an important source of pain. Histamine is released from mast cells under inflammatory conditions and evokes the pain sensation in vivo, but the cellular mechanism of histamine-induced pain has not been well understood. In the present study, we examined the effects of histamine on [Ca 2+] i and membrane potential responses to acid in isolated mouse dorsal root ganglion (DRG) neurons. In capsaicin-sensitive DRG neurons from wild-type mice, acid (>pH 5.0) evoked [Ca 2+] i increases, but not in DRG neurons from transient receptor potential V1 (TRPV1) (−/−) mice. Regardless of isolectin GS-IB4 (IB4)-staining, histamine potentiated [Ca 2+] i responses to acid (≥pH 6.0) that were mediated by TRPV1 activation. Histamine increased membrane depolarization induced by acid and evoked spike discharges. RT-PCR indicated the expression of all four histamine receptors (H1R, H2R, H3R, H4R) in mouse DRG. The potentiating effect of histamine was mimicked by an H1R agonist, but not H2R-H4R agonists and was inhibited only by an H1R antagonist. Histamine failed to potentiate the [Ca 2+] i response to acid in the presence of inhibitors for phospholipase C (PLC) and protein kinase C (PKC). A lipoxygenase inhibitor and protein kinase A inhibitor did not affect the potentiating effects of histamine. Carrageenan and complete Freund's adjuvant produced inflammatory hyperalgesia, but these inflammatory conditions did not change the potentiating effects of histamine in DRG neurons. The present results suggest that histamine sensitizes acid-induced responses through TRPV1 activation via H1R coupled with PLC/PKC pathways, the action of which may be involved in the generation of inflammatory pain.

Acute p38-Mediated Modulation of Tetrodotoxin-Resistant Sodium Channels in Mouse Sensory Neurons by Tumor Necrosis Factor-α

Tumor necrosis factor-alpha (TNFalpha) is a proinflammatory cytokine involved in the development and maintenance of inflammatory and neuropathic pain conditions. TNFalpha can have long-lasting effects by regulating the expression of a variety of inflammatory mediators, including other cytokines and TNFalpha itself. However, the speed with which TNFalpha induces tactile and thermal hypersensitivity suggests that transcriptional regulation cannot fully account for its sensitizing effects, and some recent findings suggest that TNFalpha may act directly on primary afferent neurons to induce pain hypersensitivity. In the present study, we show that peripheral administration of TNFalpha induces thermal hypersensitivity in wild-type mice but not in transient receptor potential vanilloid receptor TRPV1(-/-) mice. In contrast, TNFalpha produced equivalent mechanical hypersensitivity in TRPV1(-/-) mice and wild-type littermates, suggesting a role for TRPV1 in TNFalpha-induced thermal, but not mechanical, hypersensitivity. Because tetrodotoxin (TTX)-resistant Na+ channels are a critical site of modulation underlying mechanical hypersensitivity in inflammatory and neuropathic pain conditions, we tested the effects of TNFalpha on these channels in isolated mouse dorsal root ganglion (DRG) neurons. We report that acute application of TNFalpha rapidly enhances TTX-resistant Na+ currents in isolated DRG neurons. This potentiation of TTX-resistant currents by TNFalpha is dramatically reduced in DRG neurons from TNF receptor 1 (TNFR1) knock-out mice and is blocked by the p38 mitogen-activated protein kinase inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole]. Mechanical hypersensitivity induced by peripherally applied TNFalpha is also significantly reduced by SB202190. These results suggest that TNFalpha may induce acute peripheral mechanical sensitization by acting directly on TNFR1 in primary afferent neurons, resulting in p38-dependent modulation of TTX-resistant Na+ channels.

Low-concentration lidocaine rapidly inhibits axonal transport in cultured mouse dorsal root ganglion neurons.

Axonal transport plays a critical role in supplying materials for a variety of neuronal functions such as morphogenetic plasticity, synaptic transmission, and cell survival. In the current study, the authors investigated the effects of the analgesic agent lidocaine on axonal transport in neurites of cultured mouse dorsal root ganglion neurons. In relation to their effects, the effects of lidocaine on the growth rate of the neurite were also examined. Isolated mouse dorsal root ganglion cells were cultured for 48 h until full growth of neurites. Video-enhanced microscopy was used to observe particles transported within neurites and to measure the neurite growth during control conditions and in the presence of lidocaine. Application of 30 microM lidocaine immediately reduced the number of particles transported in anterograde and retrograde axonal directions. These effects were persistently observed during the application (26 min) and were reversed by lidocaine washout. The inhibitory effect was dose-dependent at concentrations from 0.1 to 1,000 microM (IC50 = 10 microM). In Ca2+-free extracellular medium, lidocaine failed to inhibit axonal transport. Calcium ionophore A23187 (0.1 microM) reduced axonal transport in both directions. The inhibitory effects of lidocaine and A23187 were abrogated by 10 microM KN-62, a Ca2+-calmodulin-dependent protein kinase II inhibitor. Application of such low-concentration lidocaine (30 microM) for 30 min reduced the growth rate of neurites, and this effect was also blocked by KN-62. Low-concentration lidocaine rapidly inhibits axonal transport and neurite growth via activation of calmodulin-dependent protein kinase II.

Lidocaine inhibits neurite growth in mouse dorsal root ganglion cells in culture

Recent clinical and experimental studies suggest the effectiveness of lidocaine in blocking neuropathic pain. Because it has been demonstrated that the pathogenetic mechanisms of neuropathic pain involve morphological changes in afferent neuronal terminals onto spinal cord, we examined the effects of lidocaine on neurite growth in isolated mouse dorsal root ganglion cells in culture. Incubation for 2–42 h with various concentrations of lidocaine (0.006 mM, 0.6 mM, and 30 mM) reduced the number of cells exhibiting neurites. The effects were time- and dose-dependent. Lidocaine therefore may exert its pharmacological effect, at least in part, by changing neuronal structures derived from sensory neurons.

Axonal transport is inhibited by a protein kinase C inhibitor in cultured isolated mouse dorsal root ganglion cells

We investigated roles of protein kinase C (PKC) and Ca 2+/calmodulin-dependent protein II (CAM II) kinase activities in the maintenance of axonal transport in cultured isolated mouse dorsal root ganglion (DRG) cells. Video-enhanced microscopic recordings revealed that the PKC inhibitor chelerythrine (1 μM) reduced anterograde and retrograde axonal transport, while the CAM II kinase inhibitor KN-62 (10 μM) had no effect. Morphological observation showed that neurite growth was prevented by the presence of chelerythrine (1 μM). From these results, we conclude that PKC activity is required to maintain axonal transport and thereby neurite growth.

Gradual caffeine-induced Ca2+ release in mouse dorsal root ganglion neurons is controlled by cytoplasmic and luminal Ca2+.

Cytosolic free calcium concentration ([Ca2+]c) was recorded from acutely isolated mouse dorsal root ganglion neurons loaded with Ca(2+)-indicator indo-1. The initiation of intracellular Ca2+ release by low (1-5 mM) caffeine concentrations failed to completely empty the caffeine-sensitive stores; subsequent challenge with higher doses of caffeine produced an additional [Ca2+]c elevation. This indicates a gradual Ca2+ release from caffeine-sensitive stores. The sensitivity of Ca2+ stores to caffeine was strongly influenced by endoplasmic reticulum luminal Ca2+ concentration ([Ca2+]i) as an increase in [Ca2+]i produced by a conditioning depolarization-induced Ca2+ entry, caused a several fold decrease of caffeine EC50. By elevating [Ca2+]c a threshold concentration (about 350 nM) can be reached, at which low doses of caffeine (2-5 mM) produced a regenerative Ca2+ release, that depletes the Ca2+ stores almost completely, indicating that all-or-nothing Ca(2+)-induced Ca2+ release can be generated in nerve cells.

Internalization of lectins in neuronal GERL.

Conjugates of ricin agglutinin and phytohemagglutinin with horseradish peroxidase (HRP) were used for a cytochemical study of internalization of their plasma membrane "receptors" in cultured isolated mouse dorsal root ganglion neurons. Labeling of cells with lectin-HRP was done at 4 degrees C, and internalization was performed at 37 degrees C in a culture medium free of lectin-HRP. 15-20 min after incubation at 37 degrees C, lectin-HRP receptor complexes were seen in vesicles or tubules located near the plasma membrane. After 1-3 h at 37 degrees C, lectin-HRP-receptor complexes accumulated in vesicles and tubules corresponding to acid phosphatase-rich vesicles and tubules (GERL) at the trans aspect of the Golgi apparatus. A few coated vesicles and probably some dense bodies contained HRP after 3-6 h of incubation at 37 degrees C. Soluble HRP was not endocytosed under the conditions of this experiment or when it was present in the incubation medium at 37 degrees C. Internalization of lectin-HRP-receptor conjugates was decreased or inhibited by mitochondrial respiration inhibitors but not by cytochalasin B or colchicine. These studies indicate that lectin-labeled plasma membrane moieties of neurons are endocytosed primarily in elements of GERL.