Root Ganglion Research Articles

Developmental expression of acetyl- and butyrylcholinesterase in the rat: enzyme and mRNA levels in embryonic dorsal root ganglia

Dorsal root ganglia (DRG) in the adult rat contain acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), enzymes implicated in neural morphogenesis. We used quantitative histochemistry, reverse transcription-PCR (RT-PCR), and in situ hybridization histochemistry to study cholinesterase expression during embryogenesis. Longitudinal sections of rat embryos, embryonic day 9 (E9), E11–E17, and E19, were studied by video microscopy of the stained enzyme reaction products. Both enzymes were detectable in the early DRG (E11–E12), with BChE being most prominent. There was a spatiotemporal change in expression of each cholinesterase within the DRG. From E13 on, AChE expression predominated, especially in the neuronal cell bodies, while BChE was more highly expressed in the surrounding neuropil and the ganglionic roots. This distribution resembled the pattern in adult DRG. AChE mRNA levels, as determined by RT-PCR from DRG collected at days E12–E17, and E19, varied in parallel with the intensity of enzyme stain in the DRG. Overall, these results demonstrate temporally regulated ganglionic expression of cholinesterases, which may be important in the development of the sensory nervous system.

Immunocytochemical Localization of the AMPA Receptor Subunits in the Mesencephalic Trigeminal Nucleus of the Rat

The mesencephalic trigeminal nucleus is composed of large (35–50 µm) pseudo-unipolar neurons. Closely associated with them are small (< 20 µm) multipolar neurons. An unique peculiarity of the pseudo-unipolar perikarya is that they receive synaptic input from various sources, which sets them apart from the dorsal root and cranial nerves sensory ganglia neurons. Whereas glutamate is the best neurotransmitter candidate in pseudo-unipolar neurons, glutamatergic input into them has not yet been reported. AMPA glutamate receptors are implicated in fast excitatory glutamatergic synaptic transmission. They have been localized ultrastructurally at postsynaptic sites. This study demonstrates that the pseudo-unipolar neurons of the mesencephalic trigeminal nucleus express AMPA glutamate receptor subunits, which indicates that these neurons receive glutamatergic input. Serial sections from the rostral pons and midbrain of Sprague-Dawley rats were immunostained with antibodies against C-terminus of AMPA receptor subunits: GluR1, GluR2/3, and GluR4. The immunoreaction was visualized with avidin-biotin-peroxidase/DAB for light and electron microscopy. With GluR1 antibody only the smallest multipolar neurons were recognized as immunopositive within the mesencephalic trigeminal nucleus. GluR2/3 stained the pseudo-unipolar neurons intensely within the entire rostro-caudal extent of the nucleus. In addition the former antibody stained small multipolar neurons within the mesencephalic trigeminal nucleus, though with somewhat larger dimensions than those immunoreactive for GluR1. Whereas the overall staining with GluR4 antibody! was scant, those pseudo-unipolar neurons that were stained, were strongly stained. Furthermore, a considerable number of microglial cells within and surrounding the mesencephalic trigeminal nucleus displayed very intense immunoreactivity for GluR4. These results are discussed in the light of the glutamate receptor subunit composition.

Hair follicle involvement in herpes zoster: pathway of viral spread from ganglia to skin.

Herpes zoster is caused by reactivation of varicella-zoster virus (VZV) persisting in dorsal root or trigeminal ganglia. To clarify the pathway of viral spread from the ganglia to skin, 16 biopsy specimens of early skin lesions of herpes zoster obtained from the face and trunk of 13 patients were studied histologically and immunohistochemically using monoclonal antibodies to the structural proteins of VZV. VZV-infected cells were detected in the hair follicles in 10 of the 16 specimens and in the epidermis in 2 specimens. Infected cells were localized in the isthmus of every involved follicle (12/12), frequently in the stem (8/10) and infundibulum (6/10), and never in the bulb. The high frequency of follicular involvement in herpes zoster suggests that VZV spreads to the area of skin innervated by myelinated nerves, which end around the isthmus of hair follicles and sebaceous glands.

Cranial nerves and ganglia are altered after in vitro treatment of mouse embryos with valproic acid (VPA) and 4-en-VPA

Prenatal valproic acid (VPA) exposure results in neural tube defects and in the fetal valproate syndrome (FVS), associated with developmental delay. In the present study we investigate the alterations induced by VPA and one of its metabolite, 4-en-VPA, on specific neural structures: branchial nerves and ganglia. This study was performed on 8–9 pairs of somites mouse embryos exposed in vitro for 24 h to 0.75 mM of VPA or 1 mM of 4-en-VPA. After an additional culture period of 20 h without drug, the embryos were processed for whole mount immunostaining using the monoclonal antibody 2H3, directed against the 155 kDa neurofilament protein. This technique makes it possible to visualise the branchial nerves/ganglia. VPA and 4-en-VPA induced a delay in the development of the trigeminal (V), glossopharyngeal (IX) and vagus (X) nerves/ganglia. The development of the facial (VII) nerve was delayed to a lesser extend. These treatments also induced defects in the four ganglia. The main abnormalities were a reduced dorsal component of ganglion V, the absence of the dorsal root of ganglion IX, a disorganised dorsal part of ganglion X and diffuse ventral fibres in nerves VII–VIII. In addition, scattered fibres were observed around and between ganglia. In conclusion, VPA and 4-en-VPA deeply altered the differentiation of branchial nerves/ganglia. The dorsal part of the ganglia, arising from the rhombencephalic neural crest, was particularly sensitive. The disorganisation of fibres could possibly be explained by alteration of the extracellular matrix.

Cellular localization of dopamine D2 receptor messenger RNA in the rat trigeminal ganglion.

The actions of dopamine are mediated by specific, high-affinity, G protein-coupled receptors. Multiple subtypes of dopamine receptors have been characterized, including the D2 subtype (D2R). Cells within the dorsal root and petrosal ganglia of the rat express D2R messenger RNA (mRNA) consistent with D2R expression by primary sensory neurons. We hypothesized that neurons of the trigeminal ganglion express D2R mRNA. Total cellular RNA from rat trigeminal ganglia was analyzed on Northern blots under high stringency conditions. Hybridization of trigeminal ganglion RNA resulted in a signal which comigrated with striatal, pituitary, and hypothalamic D2R mRNA. To determine the distribution of D2R expressing cells in the trigeminal ganglion, cryostat sections were analyzed by in situ hybridization followed by emulsion autoradiography. We identified a population of clustered cells labeled with dense grain concentrations over their cytoplasms. These findings demonstrate the expression of D2 dopamine receptor mRNA in discrete subpopulations of neurons in the rat trigeminal ganglion. Our observations suggest that drugs active at dopamine receptors of the D2 subtype are potential modulators of sensory activity of neurons whose cell bodies reside in the trigeminal ganglion. D2 dopamine receptors may thus have a role in clinical pain syndromes involving the head and neck.

Nitric oxide synthase-containing neurons in rat parasympathetic, sympathetic and sensory ganglia: a comparative study

In rats, the distribution of nerve structures staining for NADPH-diaphorase, and showing immunoreactivities for nitric oxide synthase (NOS), tyrosine hydroxylase and various neuropeptides was studied in sensory ganglia (dorsal root, nodose and trigeminal ganglia), in sympathetic ganglia (superior cervical, stellate, coeliac-superior and inferior mesenteric ganglia), parasympathetic ganglia (sphenopalatine, submandibular, sublingual and otic ganglia), and in the mixed parasympathetic/sympathetic ganglia (major pelvic ganglia). The coincidence of neuronal cell bodies with strong NOS-immunoreactivity and strong NADPH diaphorase reactivity was almost total. The relative proportions of NOS-immunoreactive nerve cell bodies were largest in parasympathetic ganglia and major pelvic ganglia followed by sensory ganglia. In sympathetic ganglia no NOS-immunoreactive neuronal cell bodies could be detected. In parasympathetic and major pelvic ganglia, there was a very significant neuronal co-localization of immunoreactivities for NOS and vasoactive intestinal polypeptide (VIP). This was almost total in major pelvic ganglia, in which NOS-/VIP-immunoreactive nerve cell bodies were separate from sympathetic (tyrosine hydroxylase-/neuropeptide Y-immunoreactive), suggesting that NOS-/VIP-immuno-reactive neurons might also be parasympathetic.

An opioid binding protein is specifically down-regulated by chronic morphine treatment in dorsal root and trigeminal ganglia.

Despite the recent cloning of mu, delta and kappa opioid receptors, a role in opioid receptor function for an opioid binding cell adhesion molecule is supported by several lines of evidence, including inhibition of opioid binding by opioid binding cell adhesion molecule antibodies, down-regulation of opioid binding cell adhesion molecule by chronic opioid agonist treatment of cultured NG108-15 cells, and reduction of opioid binding in NG108-15 cells by transfection of opioid binding cell adhesion molecule antisense cDNA. In the present study, we report that chronic in vivo treatment of mice with morphine results in down-regulation of opioid binding cell adhesion molecule immunoreactivity in primary afferent neurons in dorsal root and trigeminal ganglia as well as their axons. This effect was blocked by the opioid antagonist naloxone. Down-regulation of opioid binding cell adhesion molecule immunoreactivity was not observed in other areas of the central nervous system. Taken together, the previous studies which demonstrated the role played by opioid receptors in regulating release of transmitters from primary afferent neurons and the present findings of a specific regulation of opioid binding cell adhesion molecule expression by chronic exposure to morphine, provides evidence from an in vivo perspective which advances the notion that opioid binding cell adhesion molecule plays a role in the action of opioids.

Structure and innervation of longitudinal and transverse abdominal muscles of the cricket, Gryllus bimaculatus.

Detailed morphological and physiological studies on the insect abdominal muscles, including their innervation and neuromuscular transmission, are essential for understanding their important role in respiratory movements. There are both longitudinal and transverse muscles in the ventral abdominal segments of the cricket, Gryllus bimaculatus. Muscle 202 was selected as an example of a longitudinal muscle. This muscle is, on average, 1.4 mm long, paired on both sides of the abdomen, and consists of 127 fibers whose mean maximum diameter is 32 microns; the average sarcomere length is 8.1 microns. It is innervated by two ipsilateral motoneurons in the second abdominal ganglion, the axons of which run in the ipsilateral first nerve root of the third abdominal ganglion. Two motor axons run in parallel from the two cell bodies and innervate in close proximity. Accordingly, large and small excitatory junctional potentials (EJPs) are recorded from the same fiber with slightly different thresholds when the first nerve root of the third abdominal ganglion is stimulated. Muscle 203, which is a transverse muscle that extends across the fifth abdominal sternum and is located over the fourth abdominal ganglion and muscle 202 on both sides, is, on average, 2.9 mm long and consists of 86 fibers with a maximum diameter of 33 microns. The average sarcomere length is 7.9 microns. The right or left half of the muscle is innervated mainly by a contralateral motoneuron in the third abdominal ganglion through the ipsilateral first nerve root of the third abdominal ganglion. Nerve branches of the first nerve root also reach muscles 188 and 218. Muscle 203 is additionally innervated by the first nerve roots of abdominal ganglia 1, 2, and 4. These innervations were ascertained both electrophysiologically and histologically. Individual muscle fibers of muscle 203 produced small EJPs in response to stimulation of the first nerve roots of abdominal ganglia 2, 3, and 4 and large EJPs in response to stimulation of the root from the first abdominal ganglion. The large and small EJPs in muscle 203 have properties similar to those in muscle 202.

Dual excitatory innervation through the different ganglionic roots in the uropod adductor muscle of a crayfish, Procambarus clarkii

Dual excitatory innervation of the crayfish ( Procambarus clarkii) uropod adductor muscle, adductor exopodite (ADX), through different roots of the sixth abdominal ganglion, was confirmed histologically and electrophysiologically. ADX contraction induced by the excitor in the third root (R3 excitor), showed more rapid fatigue than that by the excitor in the second root (R2 excitor), possibly owing to a more rapid depletion of the transmitter substance in the process of neuromuscular transmission. The size and position of the cell bodies of the two excitors were not necessarily similar, whereas the dendritic fields appeared to be almost overlapping.

Serotonin-immunoreactive neurons and endogenous serotonin in the opisthosomal ventral nerve cord of the horseshoe crab, Limulus polyphemus.

It has been suggested that serotonin serves as a neurotransmitter in the horseshoe crab, Limulus polyphemus. While some studies of identified groups of central neurons have been conducted, little is known concerning the neuronal organization in Limulus central ganglia. This study was undertaken to determine the localization of serotoninergic neurons in the opisthosomal ventral nerve cord of Limulus and to construct a basis for further comparative biochemical and pharmacological studies of the specific function of these neurons. Endogenous serotonin was detected in the ventral nerve cord (chain of abdominal ganglia) by high-performance liquid chromatography and electrochemical detection (HPLC-EC). Endogenous serotonin was quantified in the 9th through 13th ganglia, anterior (hemal) nerves, posterior (branchial) nerves, and connectives. The serotonin content in the abdominal ganglia was significantly reduced when the ganglia were incubated for 24 hours in Leibovitz's (L-15) medium containing reserpine or 5,7-dihydroxytryptamine (5,7-DHT), neurotoxins that block the uptake of serotonin into storage vesicles. The distribution of serotonin-immunoreactive neurons in the ventral nerve cord was determined by indirect immunocytochemistry. Treatment of the chain of ganglia with an anti-serotonin antiserum followed by treatment with a fluorescent-labeled antiserum raised against the primary antibody demonstrated specific staining in each ganglion, the ganglionic roots, and connectives. Clusters of serotonin-immunoreactive neurons were observed anteriolaterally and posteriorly in each ganglion. Processes from dense fiber bundles extended from these clusters of neurons to the central region of each ganglion. These results demonstrate that serotonin-immunoreactive neurons are present in the opisthosomal ventral nerve cord of the horseshoe crab and that serotonin may function as a neurotransmitter.

Beta,beta'-Iminodipropionitrile toxicity in normal and congenitally neurofilament-deficient Japanese quails.

Morphological effects of a neurotoxin, beta,beta'-iminodipropionitrile (IDPN) were analyzed in normal and congenitally neurofilament (NF)-deficient Japanese quails. These quails (6 weeks old) were injected intraperitoneally with IDPN (0.2 g/kg body weight) three times every 3 days. They were necropsied at 10 to 12 days after the first injection. In normal quails, axonal swellings were observed histologically in the ventral motoneurons, ventral root, commissura grisea and spinal ganglion in the cervical and synsacral spinal cord. Electron microscopically, the changes consisted of increased NFs, with scattered mitochondria, smooth endoplasmic reticulum and microtubules. The myelin sheaths of the involved nerves were thinner than those of the normal axons. These lesions were similar to those induced by IDPN intoxication in mammalian experimental animals. In NF-deficient quails injected with IDPN, no axonal changes were detected. These findings suggested that IDPN selectively attacked the NFs.

Anti-GABA antibodies label a subpopulation of chemical synapses which modulate an electrical synapse in crayfish

Antibodies raised against gamma-aminobutyric acid (GABA) were used to stain sections from the crayfish abdominal nervous system, and the sections were examined under the electron microscope using a protein-A/gold conjugate secondary label. Sections were taken through the third ganglionic root, and through the interganglionic connective at the base of the third root posterior to the ganglia. The third root contains two very large motor axons, a non-GABAergic excitor (Motor Giant; MoG), and a GABAergic inhibitor (Flexor Inhibitor; FI). Only one of the two large axons stained positively for GABA, confirming that the antibody has high specificity for GABAergic neurones. The MoG is driven by powerful electrical synapses from the giant fibres, but also receives inhibitory chemical synaptic input which can gate the excitatory input. There is no physiological evidence for any other form of chemical input. However, at the ultrastructural level, the MoG is postsynaptic to three types of chemical profiles; SE-type containing round agranular vesicles, SI-type containing pleomorphic vesicles, and SM-type containing a mixture of round agranular and dense-cored vesicles. There is a highly differentiated staining pattern of these three synaptic types. Only the SI-type profiles stain positively with the GABA antibody, while the SE- and SM-type do not show significant staining. This suggests that the MoG can under some circumstances receive chemical input other than GABAergic inhibitory input. These other types of input have yet to be physiologically identified.

Synaptophysin immunoreactivity is expressed in all sensory neurones of the dorsal root and trigeminal ganglia in rat and pig

Synaptophysin immunoreactivity is expressed in all sensory neurones of the dorsal root and trigeminal ganglia in rat and pig

QUANTITATIVE EVALUATION OF NEURITE OUTGROWTH AND NEURONAL SURVIVAL OF CULTURED NERVE CELLS BY ENZYME IMMUNOASSAY AND ITS APPLICATION

Neuronal survival and neurite elongation in tissue culture are usually evaluated by counting the number of nerve cells and measuring neurite length on photographs, respectively. Because this is very time-consuming and not free of some artificial aberration, more reliable and simpler methods are needed. In this report, enzyme immunoassay (EIA) for quantitation of neurofilament protein is demonstrated to be an excellent method for evaluating neurite outgrowth and the number of surviving neurons in tissue culture. EIA is applied to the evaluation of nerve growth factor (NGF) -dependent neurite regeneration capacity in aged animals and to a study of the cytotoxic effects of dopa and dopamine on cultured nerve cells.Dorsal root ganglion (DRG) neurons were obtained from 10-week-old (Group Y), 72-week-old (Group M) and 114-week-old (Group O) male mice. Dissociated neuronal cells from each group were cultured for 4 days in chemically defined serum-free medium including various levels of NGF up to 160 ng/ml. Then EIA was performed using rabbit anti-neurofilament protein (NF) serum as the first antibody. NF content was expressed as optical density.EIA revealed less neurite outgrowth in Group M than in Group Y, but acceleration after the addition of a large amount of NGF. Neurite elongation capacity in Group O was moderately reduced at all levels of NGF when compared with the other groups. At the same time, however, significant NGF-dependent neurite regeneration capacity was preserved in aged animals. In Group Y and M, maximum length (ML : length of the longest neurite of an individual neuron) and total length (TL : sum of the length of all neurites of the neuron) were also quantified by tracing the distance on photographs. Optical densities were well correlated with ML in both groups and TL in Group Y. There was no correlation between optical densities and TL in Group M because of considerable fluctuation in TL.EIA optical density, i.e., NF content, was determined to be a reliable index for evaluating the number of surviving neurons in tissue culture. DRG neurons were obtained from 5-week-old mice and were seeded at various densities. The number of neurons counted and EIA optical density were significantly correlated. Using this method the effects of dopa and dopamine on the DRG neurons were tested. Neurons were destroyed by exposure to 0.5 mM dopa or 1.0 mM dopamine with or without superoxide dismutase and catalase, but they were spared by pretreatment with 1.0 mM deferoxamine mesylate. These results indicate that dopa and dopamine cause neuronal cell death in the presence of a small amount of iron in the culture with little or no participation by reactive oxygen species. As reported elsewhere, formation of malondialdehyde is also observed in the reaction between dopa and dissociated cerebral cortical neurons only when iron is present, which suggests that the complex of dopa (or dopamine) and iron initiates cell membrane lipid peroxidation leading to the neuronal destruction. In Parkinson's disease the mechanism of cytotoxicity may involve neuronal degeneration in the substantia nigra, which has an abundance of both iron and dopaminergic neurons. Further study is required to elucidate what triggers iron and dopa (dopamine) -mediated lipoxigenation of cell membranes in human brain in the presence of protective factors such as iron-transporters, tocopherols, phosphates, etc.

An identified cell is required for the formation of a major nerve during embryogenesis in the leech.

Investigations of the cues by which axonal growth cones navigate long distances to their targets have revealed the use of a rich and complex diversity of cellular and extracellular information. In the present study we describe one of the most conceptually simple pathfinding cues: a single identified cell in the leech, Hirudo medicinalis, that may guide axons several hundred micrometers to innervate a particular target. One of the stereotyped nerves of H. medicinalis is a "sex nerve" that projects from the anterior root of ganglion 6 [SNA (6)] to the male reproductive structures in the adjacent anterior segment. The pathway for SNA (6) is completely underlain by a single peripheral cell, here called the axonal runway cell (ARC), before axons enter the pathway. The ARC is apparently a nonneuronal cell that stains with a monoclonal antibody that recognizes leech muscle cells. The importance of the ARC for establishing SNA(6) was tested by ablating it before axons entered the pathway. When the ARC was killed either by physical disruption with a microelectrode, or by photoablation after filling it with the fluorescent dye Lucifer yellow, SNA(6) always failed to form, whereas all other nerves formed normally. Killing other peripheral cells in proximity to the ARC did not interfere with SNA(6) formation. Ablation of possible "pioneer neurons" for SNA(6) also did not prevent its formation. These results show that formation of a particular nerve requires only a single cell to serve as a guide for outgrowing processes.

Plasminogen activator secretion in relation to Schwann cell activities

The molecular characterization of neural secretory plasminogen activators and their possible association with two well-defined Schwann cell activities (mitosis and migration) were investigated. Schwann cells from peripheral sensory ganglion roots were established as primary cultures of high purity. Conditioned culture medium was fractionated by gel electrophoresis and assayed for plasminogendependent in situ caseinolytic activity. Both tissue-type and urokinase-type plasminogen activators were detected early in culture. The amount of the tissue-type form decreased with increasing time in culture. Proliferative ability measured as [3H]thymidine incorporation into the nucleus varied under several culture conditions and was lowest in defined serum-free medium; however, these serum-free cultures expressed the highest level of plasminogen activator secreting cells. Combined autoradiography/fibrin overlay assays allowed for a direct analysis at the single cell level; interestingly the findings did not support the idea of an obligatory expression of the enzyme in replicating cells. Cell motility was assayed in combined gold particle clearing/fibrin overlay assays and likewise no obligatory association of protease secretion and cell movement could be demonstrated. The results are discussed in relation to previous tudies in this field.

Neurokinin A and Galanin in the Thyroid Gland: Neuronal Localization*

The distribution of neurokinin A (NKA) and galanin (GAL) in the thyroid gland of several species was examined with immunocytochemistry. NKA-immunoreactive fibers were observed around blood vessels and follicles in all species examined, whereas GAL-immunoreactive fibers were found in mice and rats only. NKA-containing fibers were more numerous than GAL-containing fibers. All thyroid NKA-containing fibers harbored substance P (SP), and the majority of them stored calcitonin gene-related peptide (CGRP) as well. Most thyroid GAL-immunoreactive fibers contained NKA, SP, and CGRP; in a minor population GAL coexisted instead with vasoactive intestinal peptide. Cervical vagotomy (extirpation of the nodose ganglion) reduced the number of NKA- and GAL-containing fibers in the thyroid by approximately 50%. The jugular ganglion and cervical dorsal root ganglia are fairly rich in GAL-, NKA/SP-, and CGRP-containing cell bodies, which presumably represent the source of GAL-, NKA/SP-, and CGRP-containing fibers in the thyroid. The thyroid ganglion is rich in vasoactive intestinal peptide nerve cell bodies, which presumably project to the thyroid gland; a minor proportion of these cell bodies was found to contain GAL as well. Although the distribution of NKA and GAL fibers in the thyroid suggests that the two peptides are involved in the regulation of local blood flow and follicular cell activity, neither NKA nor GAL had any influence on thyroid hormone release as tested in conscious mice.

Response to axotomy of an identified leech neuron, in vivo and in culture

Membrane properties of identified leech neurons with non-spiking somata were studied after axotomy. Two distinct procedures were used: the section of ganglionic roots in vivo and the isolation of cell somata in culture. The results indicated that axotomized neurons progressively developed the excitability of somatic membrane, both in vivo and in culture.

Herpes simplex virus (HSV) infections.

This infection is caused by reactivation of varicella-zoster virus (VZV), which may remain latent in the dorsal root and cranial nerve ganglia for decades. Reactivation often occurs for no apparent reason, although stress and immunosuppression may increase the risk.

Segmental giant: evidence for a driver neuron interposed between command and motor neurons in the crayfish escape system.

1. The giant command neurons for tailflip escape behavior in crayfish have been thought to excite the nongiant fast flexor (tailflip producing) motor neurons (FFs) via monosynaptic connections. We show here that excitation of FFs instead occurs via a bilateral pair of segmental giant neurons (SGs) interposed between the command axons and FFs in each segment. 2. Anatomically, the SGs appear to make numerous contacts with ipsilateral command axons and FFs and fewer contacts contralaterally. In contrast, the command axons have only sparse direct connections to the FFs. An SG has an axon in the ipsilateral first ganglionic root and may be a modified swimmeret motor neuron. 3. Each SG is depolarized well beyond threshold by the firing of an ipsilateral command axon and is depolarized to near threshold by the firing of a contralateral command axon. The synapses between command axons and SGs are electrical and probably rectifying. 4. Each FF is excited to a level near firing threshold by the SG ipsilateral to its axon and is excited weakly by the contralateral SG. The synapses between SGs and FFs are electrical and nonrectifying. 5. Variations in excitatory postsynaptic potentials (EPSPs) recorded in FFs during prolonged, high-frequency firing of the command axons can be accounted for by refractoriness of SG spikes, as opposed to refractoriness of dendritic branch spikes as had previously been delivered. 6. These findings illustrate the limitations of conventional tests for monosynapticity. 7. The functional significance of having driver neurons interposed between command neurons and motor neurons is discussed.