Histologic correlation of the degenerating facial nerve with electroneurography.

The myogenic compound action potential (CAP) measured by electroneurography (EnoG) is hypothesized to correlate directly with the number of viable motoneurons in the facial nerve. In an animal model (cat), two independent ENoG techniques, standardized recording lead placement (SRLP) and optimized recording lead placement (ORLP), were used to record CAPs evoked from nerves undergoing degeneration. Normal test-retest variability simulated human studies. Peripheral counts of viable axons correlated with N1 amplitude (ORLP ENoG), peak-to-peak (absolute) amplitude (both ENoG techniques), and area under the negative phase of the diphasic CAP waveform (both ENoG techniques) recorded over the whisker pad. These results validate ENoG as a reliable indicator of neural integrity following traumatic lesions of the facial nerve.

Accumulation of neurofilament proteins in the regenerating facial nerve.

In order to compare the extent of axonal regeneration in two surgical nerve repair procedures, we measured the levels of the neurofilament (NF) proteins in the regenerating facial nerve of adult New Zealand rabbits. The animals were operated on bilaterally, with a chamber model placed on one side and a cable graft model inserted on the contralateral side. Normal nerve from unoperated animals or nerve removed during nerve repair surgery served as controls. Using immunoblot techniques and densitometric measurement, we examined specific changes in the individual NF [High (H), Medium (M), and Low (L) molecular weight (MW)] in the regenerating nerve at 3 and 7 weeks postoperation time. Linearity of the densitometric system was established by separation of serial dilutions of known NF on the gel, and blotting for immunostaining. The amount of all 3 NF's decreased during the regeneration process compared to normal nerve, but there were differences between the two procedures. The NFH in the distal segment of the chamber repaired nerve at 7 weeks was 60-70% of the preoperative state, which correlated with a previous morphological study of axona) caliber during regeneration. At 3 weeks, NF content was lower in the distal segment of the chamber than in the distal cable graft. By 7 weeks, NF content was similar in proximal and distal segments of both models. Thus, although initial regeneration is slower in the chamber model, the eventual outcome is similar in both chamber and graft.

Targeted neuronal lesion induced by photosensitizing dyes.

Free radical-induced phototoxicity mediated by laser irradiation was investigated in the rabbit facial nerve. Azure-C, mesoporphyrin, or the dye conjugated to the protein carrier horseradish peroxidase were injected into the levator alae nasi muscle. Two to 7 days after uptake and laser exposure, nerve sections showed varying degrees of cellular modifications including: severe membrane degradation and associated lipid peroxide granules, distended mitochondria, and mitochondrial loss. Immunoblots of homogenates from treated nerves revealed specific changes in neurofilament and myelin basic protein. The site specific damage produced in vivo by photosensitizing dye resembles abnormalities in aging neurons and in Batten's disease, both hypothesized to be cases of free radical-peroxidation reactions. These reactions differ from those found in transection and crush lesions.

Stimulation variables in electroneurography of the facial nerve.

Electroneurography is an electrophysiologic measurement of compound action potentials elicited by the supramaximal stimulation of a peripheral nerve. The stimulus intensity-response function was examined in the cat facial nerve following stimulation over two different sites of the facial nerve: the main trunk and the buccal branches. Thresholds were achieved at lower current settings, the resultant compound action potential exhibited a greater amplitude, and masseter muscle excitation was more evident following stimulation over the buccal region. The use of concurrent facial- and masseter-evoked electromyographic monitoring allows more sensitive detection of masseter excitation and a more accurate determination of the current level necessary for supramaximal stimulation.

Analysis of axonal regeneration through the silicone regeneration chamber: a retrograde tracing study in the rabbit facial nerve.

Transected facial nerve buccal branches in the adult rabbit were sutured to a silicone growth chamber and regeneration was observed at 3, 5, and 7 weeks postoperation. Using the retrograde axonal transport of horseradish peroxidase technique the soma in the facial motor nucleus were counted and the number was correlated with the number of axons found in the mid-cross section of the regenerating buccal branch and with the number of axons in the original nerve. The somatotopic reorganization in the facial motor nucleus was examined. The mean number of facial motoneuron soma labeled with HRP in the control was 68.0% (+/- 18.6, SE) of the axons counted at periphery. In the regenerating nerves, the labeled soma represented 2.4% of the preoperative controls after 3 weeks and rose to 8.6 and 43.9% at 5 and 7 weeks, respectively. At 3 weeks postoperative time, four of six regenerating nerves did not contain any myelinated axons at the center cross section. The ratios of labeled soma to the regenerating myelinated axons counted at the center cross section at 5- and 7-week time points were 42.2 and 61.1%, respectively. The location of the soma found in the early regeneration phase was similar to the normal distribution except in the dorsal subnucleus. After 7 weeks the proportion of labeled soma in the intermediate subnucleus declined but the general pattern replicated the distribution found in normal control preparations.

Acetylcholine in the crayfish optic lobe: concentration profile and cellular localization.

The crayfish optic lobe contains high levels of acetylcholine (ACh) and choline as measured with a chemiluminescent assay in small fragments of optic lobe tissue. The highest concentrations were found in the medulla externa and medulla interna (second and third optic neuromeres), which have ACh concentrations of 270 pmol/mg tissue. This concentration is about 16 times that measured in the photoreceptors and lamina ganglionaris (the first optic neuromere). Immunocytochemistry (based upon antisera to choline-glutaryl-BSA) revealed low levels of ACh-like reactivity in the lamina ganglionaris associated with the terminal arbors of centrifugal and/or tangential neurons. The most intense ACh-like reactivity was observed in monopolar neurons of the medulla externa and medulla interna. One monopolar neuron/medullary column (or about 2500 neurons/medullary neuropile) exhibited reactivity and an estimated cytoplasmic concentration of 8.1 mM.

Facial nerve regeneration in the silicone chamber: the influence of nerve growth factor.

The role of nerve growth factor (NGF) was examined in the neural repair of adult rabbit facial nerves using an in vivo preparation. A 35-microliters nerve growth chamber was created by suturing the proximal and distal ends of a transected facial nerve (superior buccal branch) into a silicone tube. A gap of 8 mm in the chamber remained after removal of a 5-mm piece of nerve and insertion of the proximal and distal stumps into the tube. Animals were operated bilaterally; one side of the chamber was filled with NGF and the contralateral side was filled with Ringer's solution. Regeneration of the nerves was examined 1 to 5 weeks following the surgery. The caliber of the nerve bundle, the distribution pattern of regenerating motoneurons, axon number per fascicle, size distribution, and the total number of cells were compared to the preoperative morphology pattern found for that animal. Each buccal branch served as its own control. The NGF-filled chambers demonstrated an overall larger caliber of nerve regeneration at 5 weeks and a higher density distribution of axon growth at 3 and 5 weeks. In the early regeneration case (3 weeks), the axon growth profile exhibited more fascicles and less axons than the preoperative controls. In the more advanced state (5 weeks), the fascicle number was reduced and the axon number was increased. After 5 weeks of regeneration the number of fascicles was still more than that found in the preoperative state. Axon size at 5 weeks was 80% that of the preoperative controls and the thickness of the myelin sheath was less than the preoperative level. The histogram of the size distribution revealed the same distribution as in the preoperative control section.

Immunocytochemical studies of the distribution of acetylcholine in the crayfish brain.

A number of studies indicate that acetylcholine is an important transmitter in most crustacean primary afferents and in at least several central pathways. Little is known, however, regarding the structure or distribution of cholinergic pathways in the central nervous system. The recent introduction of antibodies to choline-protein conjugates provides a potentially powerful means for localizing putative cholinergic neurons and pathways in the nervous system. Acetylcholine was localized with immunocytochemical procedures in the axons and terminals of cephalic primary afferents and in interneurons of the crayfish brain. The most intensely reactive loci were the primary sensory neuropiles, which contain the terminals of the statocyst afferents (parolfactory lobes) and antennal afferents (antennal lobe). These results are generally in accord with previous findings based upon choline uptake and enzyme assay in lobster cephalic nerves. We also found evidence consistent with the presence of acetylcholine in the globular interneurons of the accessory lobe and in descending interneurons which originate in the dorsal medial and anterior clusters of the protocerebrum. The axons of several neurons in the circumesophageal connective (descending interneurons and primary afferents) are also reactive to the choline antibody.

The functional organization of the crayfish lamina ganglionaris. II. Large-field spiking and nonspiking cells.

The functional properties of the multicolumnar interneurons of the crayfish lamina ganglionaris were examined by intracellular recording and the cell structures were revealed with the aid of Lucifer yellow or horseradish peroxidase iontophoresis. The multicolumnar monopolar cell M5 responds to a light pulse with a depolarizing compound EPSP and a burst of action potentials. Both the EPSP amplitude and the spike rate decay toward a lower level plateau in less than 200 ms after light onset. M5 is subject to surround inhibition, which is associated with a compound IPSP and net hyperpolarization of the membrane potential. Direct depolarization of M5 may provide a weak excitatory drive to medullary sustaining fibers (SF). Tangenital-cell type 1 (Tan1) has a broad expanse of neurites in the lamina (covering 10 to 15 cartridges) and a much narrower projection in the medulla (1 to 3 cartridges). The response to a light pulse has a long latency consistent with a polysynaptic receptor to Tan1 pathway. The response consists of a nearly rectangular hyperpolarization. Light 'off' elicits a depolarization and a burst of impulses. The polarity of the 'on' response can be reversed by hyperpolarizing the membrane by 23 mV. The receptive field is broad and the intensity-response function exceeds 4 log units. Direct hyperpolarization of Tan1 provides a strong excitatory signal to medullary SFs both in the dark and in the presence of illumination. We propose that Tan1 provides the principal steady-state excitatory drive to the SFs. Tangential-cell type 2 (Tan2) is distinguished from Tan1 by the extent and shape of the lamina process, which is a vertically oriented neurite spanning most of the lamina in a single plane. Functionally, Tan2 is similar in most respects to Tan1, but the response latency is much shorter, comparable to that of monopolar cells. T-cells may exhibit spontaneous impulse activity in the dark which is inhibited by a short latency hyperpolarizing light response. The receptive field, which is about 2 X larger than that of the columnar monopolar cells, is correlated with a small but multicolumnar dendritic arbor in the lamina. Since T-cells are aminergic, it is possible that the amines are normally released in the dark. A single amacrine cell was fully characterized. It exhibited a short latency hyperpolarizing response to light onset and a strong depolarizing 'off' response.(ABSTRACT TRUNCATED AT 400 WORDS)

The functional organization of the crayfish lamina ganglionaris. I. Nonspiking monopolar cells.

The light responses of the second order lamina monopolar neurons were examined in the crayfish compound eye. Single cartridge monopolar neurons (M1-M4) exhibited nonspiking hyperpolarizing light responses; for M1, M3 and M4 the transient 'on' response operated over the same intensity range as the receptor, 3.5 log units. M2 operated in a much narrower intensity range (1.5 log unit). The 'on' responses were associated with a 19% increase in conductance. The hyperpolarizing 'on' response can be reversed at 18 mV below the resting membrane potential. The half-angular sensitivity width of monopolar cells (in partially dark-adapted eyes) is 15 degrees X 8 degrees (horizontal by vertical). Off axis stimuli elicit attenuated hyperpolarizing responses associated with a diminished conductance increase or depolarizing responses associated with a net decrease in conductance. The latter result is consistent with the presynaptic inhibition of a 'back-ground' transmitter release which normally persists in the dark. Lateral inhibition is elicited from the area immediately surrounding the excitatory field, and it is associated with diminished transient responses and an accelerated decay of the response. Inhibitory stimuli decrease the conductance change associated with the hyperpolarizing response. The surround stimuli can also elicit depolarizing 'off' responses with reversal potentials positive to the membrane resting potential. It is concluded that the rapidly repolarizing monopolar cell response is modulated by both pre- and postsynaptic inhibitory mechanisms. A compartment model indicates that signal attenuation along a 500 microns length of monopolar cell axon is 22-34%. Simulation of steady-state signal transmission suggests that passive (decremental) conduction is sufficient to convey 66 to 78% of the monopolar cell signal from lamina to medulla. The current-voltage relation in current clamp is linear over the physiological operating range, and there is no evidence for rectification. Hyperpolarization of single monopolar cells (M1-M4) provides a polysynaptic excitatory signal to the medullary sustaining fibers.

Integration and spike initiation in neuronal terminals.

Axoaxonal synapses impinge on the terminals of neurons and are thought to play a crucial role in the regulation of synaptic transmission. In the crayfish brain, the terminals of several identified interneurons exhibit both EPSPs and IPSPs in response to cephalic sensory stimulation. The terminal arbors extend into primary sensory neuropils near the terminals of primary afferent axons of the antennae and statocysts. The EPSPs arise monosynaptically from primary afferent stimulation and can elicit action potentials. Bidirectional conduction is routinely observed. Terminal IPSPs are transmitted via a polysynaptic pathway. Furthermore, two morphological classes of synaptic profiles impinge on the presynaptic terminals. These results indicate that the neuron terminal can function as a second, independent site of synaptic integration.