This lab investigated human reflexes, particularly of the monosynaptic stretch reflex of the gastrocnemius via electrical tibial nerve stimulation and the polysynaptic vestibulo-ocular reflex. A reflex can be defined as an involuntary and very rapid movement generated in response to some sort of stimulus; the latency period is the amount of time it takes for the stimulus to create a response. The stronger the stimulus, the stronger the response will be.
Reflexes occur via the reflex arc which is composed of five central components. A stimulus causes the muscle’s receptors, the muscle spindle which is a modified muscle fiber containing its own afferent and efferent nerve fibers, to stretch which propagates the signal to the IA afferent neurons (sensory neurons to the spinal cord). Once the afferent limb has been stimulated, the signal then enters the spinal column through the dorsal root. This region is called the integrator as it integrates the response from sensory neurons to the spinal cord and then the synapses with the efferent alpha motor neurons in the ventral horn which become excited. The stimulus proceeds to exit the spinal column through the ventral root and causes muscle contraction (Misiaszek, 2003, p. 144).
A monosynaptic reflex is the simplest reflex as one afferent neuron synapses directly onto another efferent neuron. The Hoffman Reflex (H-Reflex) is an electrically induced monosynaptic reflex that was studied through stimulation of the tibial nerve containing both IA afferents and alpha motor neurons. The goals of this section of the lab were to study the response of the H-wave and M-wave as the stimulus intensity and stimulus frequency were increased. These are produced by recording electromyograms (EMGs) via electrodes that are hooked up to the non-athletic, slender-legged female subject and tell the sum of the electrical activity in the muscles at that point. Raising stimulus intensity should result in a gradual decrease in the size of the H-wave (after H-wave threshold is reached) although the M-wave will occur before the H-wave (once its threshold is reached) and increase in size as the voltage goes up since it has a shorter latency period (Palmieri et al., 2004, p. 269). Increasing stimulus frequency at low voltages should leave the M-wave unaffected, but the H-wave will decrease in amplitude because the Renshaw cells, which are inhibitory interneurons that act as a negative feedback mechanism, release glycine to create an overlap between stimulus frequency and hyperpolarization caused by the Renshaw Cells (Nishimura et al., 2010, p. 3437).
The vestibulo-ocular reflex is a complex polysynaptic reflex, a pathway with one or more interneurons connecting afferent and efferent neurons, which responds to angular acceleration of the head and dictates eye movements. The sensory structures are the three semicircular canals with one set in the x, y and z planes of each ear. The receptors are hair cells that are located within a gelatinous mass called the cupula and project into the viscous endolymph inside the semicircular canals. When the shorter stereocilia hairs move towards the one long kinocilium hair, depolarization occurs and the vestibular nerve is activated to respond. When the subject is spun, they will experience nystagmus which is alternating slow and fast components of eye movement in response to quick changes in acceleration. With open eyes at slow speeds, fast eye movement should be in the direction of the spin while the slow eye movement should be in the opposite direction and the fast eye movement should be in the direction of perceived motion when the subject closes his eyes and is abruptly stopped.
Materials and Methods
Part 1: The non-athletic, slender-legged 21 year old female subject laid down on a table and three electrodes were attached to her right leg after proper cleaning; one on the gastrocnemius muscle, one a little higher on the same muscle and one on the Achilles tendon as a baseline to reduce electrical noise. The stimulating electrode was taped to the popliteal space. The electrodes sent signals that were shown as EMG recordings. For a more detailed protocol, refer to NPB 101L Systemic Physiology Lab Manual (Bautista et al., 2008, p. 19-29).
Stimulus Voltage: Stimulus voltage was gradually increased to see and record the appearance and disappearance of the H-wave and the appearance of the M-wave.
Stimulus Intensity: The stimulus intensity was adjusted to .5, 1, 2 and 3 Hz at the intermediate H-wave voltage to record the appearance of the H-wave and M-wave.
Part 2: Vestibular Apparatus: A different 21 year old male subject sits in a chair in all four cases
Case 1: Subject’s head is tilted forward 30° and is spun clockwise with closed eyes 10 times in 20 seconds. Subject’s thumbs indicate perceived direction of movement (up means no rotation).
Case 2: Open-eyed subject is rotated clockwise 1 turn/minute.
Case 3: Close eyes and tilt head forward 30° and rotate clockwise 1 rev/sec for 10-15 sec and stop chair and tell subject to keep eyes open and stare ahead. Rest for 3-5 minutes.
Case 4: Lean head over left shoulder, close eyes and repeat rotation steps in Case 3. Tell subject to tilt head upright and open eyes to stare ahead.
The H-wave first appeared at 42.5 volts with a small amplitude of .472 millivolts (mV). The intermediate H-wave of 2.99 mV was seen soon after at 45 volts and shortly thereafter, the M-wave threshold was reached at 52.5 volts. The M-wave had an amplitude of 1.26 mV as the H-wave amplitude continued to increase at 52.5 volts to 3.18 mV. The H-wave reached a maximum amplitude of 3.97 mV at 54 volts and then proceeded to steadily drop until it was completely abolished at 75 volts. As shown by Figure 1, the M-wave increased all the way through the experiment (once its threshold was reached) reaching 1.36 mV at the Max H-wave threshold and jumped to 6.46 mV when the H-wave abolished.
While the H-wave was the first wave to appear, as soon as the M-wave threshold was reached, the M-wave was produced before the H-wave for the remainder of the experiment. The latency period for the H-wave was about 26 milliseconds (distance from the stimulus to the onset of the H-wave). The latency period for the M-wave was considerably shorter at about 8 milliseconds.
The effect of stimulus frequency was shown by keeping the voltage constant at 45 volts, the intermediate H-wave threshold. As shown by Figure 2, the H-wave followed a steady downward trend, dropping in amplitude. The M-wave, however, stayed pretty constant. The M-wave amplitudes at .5 Hz, 1 Hz and 3 Hz were the same at .87 mV with only a slight dip to .82 mV at 2 Hz resulting in a straight horizontal trendline. The H-wave’s amplitude dropped from 3.99 (at .5 Hz) to 3.69 (at 1 Hz) to 3.05 (at Hz) to 2.86 mV (at 3 Hz) as the frequency was increased.
In case 1 of part 2, the subject was rotated clockwise and his perceived motion was observed. The subject initially believed that his motion was in the opposite counterclockwise direction and indicated so with his thumbs facing in that direction for the first 10 seconds. After about 10 seconds, the subject had no perception of his direction of motion and indicated so by pointing his thumbs upward for about 8 seconds. At the end of the spin, he slowly changed his thumbs to point back in the counterclockwise direction as he slowed to a stop for 2 seconds. In case 2, the subject was spun very slowly with his eyes open and the subject’s slow eye movement was in the opposite direction (counterclockwise) of his clockwise rotation. His fast eye movement was in the same clockwise direction as his movement. Case 3 produced different results however when the subject closed his eyes. Once he was abruptly stopped, the subject’s eyes bounced back and forth momentarily before the fast eye movement proceeded in the opposite counterclockwise direction while the slow eye movement was clockwise; there were no noticeable changes in posture as he remained straight up. In case 4, the procedures from case 3 were repeated except the subject’s head was tilted over his left shoulder, and the subject’s eyes moved up and down rapidly in a slightly diagonal direction. Postural changes were noted as the subject leaned forward in the chair after being stopped.
My original data was incorrect so another group’s data was used for Part 1 of the experiment. My group’s data was acceptable for Part 2 (vestibulo-ocular reflex). The data provided by the group was fairly accurate with one exception. The intermediate H-wave threshold was 45 volts and the M-wave threshold was 52.5 volts, but when testing the effects of stimulus frequency in the second half of Part 1, M-waves appeared. This should not have happened as the voltage was set at 45 volts which should not have been high enough to induce an M-wave unless one of those thresholds was wrongly recorded.
The Hoffman or H-reflex is an electrically induced monosynaptic reflex that signifies the typical five step reflex arc with one exception. The receptor and muscle spindle are bypassed to stimulate the afferent neuron, but the stimulus still travels through the rest of the arc. The muscle spindle is the muscle receptor that sends the stimulus through the reflex arc and controls changes in muscle length and tension. Stretching of muscles activates the muscle spindle to also stretch and the stimulus goes through the entire reflex arc to cause contraction (Sherwood, 2008, p. 177). The H-reflex begins with a stimulus that activates the IA afferents which then synapses directly onto the alpha motor neurons in the spinal cord and this leads to muscle contraction (Palmieri et al., 2004, p. 268). This reflex arc gives rise to the H-wave. The M-wave is produced via a different mechanism called direct stimulation. Direct stimulation is simply the process of bypassing the reflex arc to directly stimulate the efferent motor neuron to produce a response.
The H-wave is the early-presenting wave. At low voltages, the stimulus travels through the entire reflex arc so only the H-wave will show up on the EMG. Only sensory neurons are activated at low voltages because they are larger than efferent motor neurons. This can be explained by Ohm’s Law V=IR where V is voltage, I is current and R is resistance. According to Ohm’s Law, if voltage is held constant, when resistance is decreased then the current flow must in turn increase. More pertinent to this lab is the fact that voltage and resistance are directly proportional (Howard, 2008). Since the 1A afferents have a larger diameter, there is less resistance through them so a weaker stimulus i.e. a lower voltage is needed to activate them. Thus when the voltage is low, the wave form that appears is the H-wave because it is easier to stimulate larger afferent neurons than smaller efferent alpha motor neurons (Palmieri et al., 2004, p. 269).
As the voltage is increased, the H-wave continued to steadily increase and around 52.5 volts, the M-wave began to appear and it appeared before the H-wave. The M-wave appeared before the H-wave as soon as the 52.5 volt M-wave threshold was reached because the alpha-motor neurons were activated directly. The M-wave is universally accepted to have a shorter latency period of only 10 to 20 milliseconds and displayed an 8 millisecond latency period in the lab. The H-wave displayed a 26 millisecond latency period which is also close to the physiologically accepted 30 millisecond latency period for the H-wave (Palmieri et al., 2004, p. 269).
The latency period changes with the location of the muscle. The M-wave appeared first at the M-wave threshold because the distance the action potential needs to travel is much shorter since the stronger stimulus does not travel through the entire reflex arc. For the M-wave, the travelled path does not include the afferent limb and the signal bypasses the spinal cord as well. As the stimulus intensity (voltage) is further increased, the M-wave still appears first and it continues to increase in amplitude while the later H-wave’s amplitude steadily declines. This is because more alpha motor neurons are directly activated and skip through the afferent and integrator region of the reflex arc so less alpha motor neurons are available for typical activation through the entire reflex arc by the H-reflex (the alpha motor neurons that are directly stimulated are different than the ones activated by the H-reflex) (Misiaszek, 2003, p. 146). Furthermore, these H-reflex alpha motor neurons are in their absolute refractory period. The absolute refractory period is the point in time when an excitable neuron becomes hyperpolarized and cannot be stimulated until a period of rest (Sherwood, 2007, p. 98). The correlation coefficient was also fairly high at .711 indicating that there is a strong correlation between stimulus intensity and amplitude of the H-wave and M-wave.
Increasing stimulus frequency had a different effect on the H-wave as exemplified by the essentially opposite trendlines for the H-wave shown in Figure 1 and Figure 2. Increasing the stimulus frequency steadily decreased the H-wave amplitude because of recurrent inhibition by Renshaw cells. Renshaw cells are monosynaptic with 1A afferent nerve fibers that synapse onto alpha motor neurons, but they act as polysynaptic neurons. While the afferent limb does directly synapse onto the efferent limb, there is still an inhibitory interneuron on the efferent that acts as a negative feedback loop (Nishimaru et al., 2010, p.3437). An excitatory stimulus from the small branches of the motor neurons leads to the activation of the inhibitory interneuron in the postsynaptic cells prompting action potentials to propagate down to the synaptic terminals. Once the action potential reaches the inhibitory interneuron synaptic terminals, the inhibitory neurotransmitter glycine is released and diffuses across the synaptic cleft to bind to a receptor site on the postsynaptic motor neurons. Glycine leads to membrane hyperpolarization in these motor neurons and inhibits them from firing and producing muscle contractions (Nishimaru et al., 2010, p. 3437). Recurrent inhibition is also a very physiologically useful process since it prevents extended periods of tetanus. It is a key system of checks and balances for the muscles. The M-wave was basically unaltered except at 2 Hz when its amplitude dropped from .87 mV to .82 mV. This is because the M-wave was not at a high enough voltage to experience this recurrent inhibition. If the voltage was set at a higher reading, then the M-wave would have also experienced a steady drop in amplitude and a refractory period due to the high frequency of input preventing the neurons from relaxing. Low frequency of firing allows for greater glycine re-uptake to prevent hyperpolarization.
The vestibulo-ocular reflex is a very complex polysynaptic reflex that allows the body to maintain equilibrium by focusing objects on the retina in response to postural and head movements. The reflex arc starts with a change in angular acceleration of the head which causes the hair cells to depolarize and activates the vestibular nerve so the signal travels up to the vestibular nuclei, a group of neuronal cells bodies in the brain stem, and to the cerebellum before the signal is integrated with multiple other parts of the body. The brain stem is the effector in this reflex arc and the efferent limb of the arc is the alpha motor neurons that send the signal to the external lateral and medial eye muscles which causes nystagmus based on the specific change in acceleration (Sherwood, 2008, p. 227). The vestibular apparatus is composed of the semicircular canals and the otolith organs which are located deep within the temporal bone of the ear next to the cochlea. The semicircular canals are composed of three planes in the x, y and z directions in each ear that are positioned orthogonally or to 90° of each other.
The vestibular apparatus contains hair cells surrounded by a gelatinous layer called the cupula within the ampulla (an enlarged canal) of the semicircular canals that respond via mechanical deformation caused by the viscous endolymph fluid. The hair cells are activated via mechanotransduction. There is one long hair cell or cilium called the kinocilium and 20-50 increasingly shorter microvilli called stereocilia that are attached together via tip links which are the key determiner of whether the Calcium (Ca2+) channels open or close. Positive mechanical deformation occurs when the shorter stereocilia are activated to bend towards the kinocilium and Potassium (K+) channels are opened in the stereocilia through which K+ ions enter leading to depolarization. This is a very unique process as depolarization in other parts of the body usually entails the influx of Sodium (Na+) ions and the efflux of K+ ions out of the cell. The hair cells are an unusual case where the concentration of K+ ions is actually higher outside of the cell than inside. Nevertheless, this depolarization results in the opening of voltage-gated Ca2+ channels enabling Ca2+ to enter the cells and vesicles to fuse with the pre-synaptic membrane and the excitatory neurotransmitter acetylcholine is released and diffuses across the synaptic cleft to bind onto receptor cells to activate the vestibular nerve. If the stereocilia bend away from the kinocilium, then the cell becomes hyperpolarized and the K+ channels close and there is no release of the excitatory neurotransmitter to induce activation of those hair cells (Sherwood, 2010, p. 218-219). It is critical to also note that when the hair cells in one ear are depolarizing, the hairs in the other ear are hyperpolarizing and vice versa. This is how motion is sensed in one direction in each plane.
Surprisingly, the subject did not point his thumbs in the direction of perceived motion at the beginning of the spin in case 1. This is somewhat odd because as a subject is turned clockwise, the endolymph fluid in the semicircular canals should slowly begin to rotate in the opposite direction (after an initial delay due to its viscosity) and the direction of perceived motion should be opposite the motion of the endolymph. In sum, the subject should have perceived his motion in the clockwise direction that he was moving. In the middle of the spin, the patient’s thumbs pointed upwards which indicated that he did not perceive any motion. The viscous endolymph takes time to catch up with the actual speed of the body since the body is still accelerating (Sherwood, 2007, p. 220). The endolymph’s speed is equal to the body’s speed once the body reaches a constant velocity and this is when the subject had no perception of motion (St George et al., 2010, p. 11-12). The physiological principle behind this phenomenon is that the stereocilia are not moving in either direction so there is no depolarization or hyperpolarization and the subject has no idea in which direction he is spinning.
The subject’s eyes are closed because if his eyes are open, he can retrace his direction of rotation. Everyone else is also told to be quiet for similar reasons. The goal is for the subject to only rely on the vestibular system to discover which way he is moving. After the chair stopped, the subject predictably pointed in the opposite counterclockwise direction because as he stopped, the endolymph fluid continues in its direction of motion and moves faster than the decelerating body. As a result, the subject will perceive motion in the opposite direction since the stereocilia are now bent in the opposite direction, counterclockwise.
In case 2, the subject’s lateral slow and fast eye movement adhered to typical physiological principles. When moving slowly clockwise, the subject’s slow eye movement was in the opposite counterclockwise direction as he attempted to keep the previous stationary object in his field of view. Eventually, he ran out of room in his field of view and could not keep the object in sight so his eyes quickly reset in the direction of movement (clockwise) and focused on a new stationary object. Therefore, the eyes were slow tracking in the opposite direction of movement, but once they moved too far laterally, they fast reset in the opposite direction to acquire a new field of vision. This nystagmus is characterized by direction of rapid eye movement. The subject will display nystagmus regardless of whether they are in the dark or close their eyes since nystagmus relies on sensory input only from the vestibular apparatus, not needing sensory input from the eyes. The eyes will always match the direction the vestibular is telling it.
In case 3, the subject’s fast eye movement was in the opposite direction of his motion after he was suddenly stopped which directly contrasts with case 2. This is because the subject cannot rely on input from his visual system; he can only rely on his vestibulo-ocular reflex and the movement of the hair cells since his eyes are closed. He had no field of vision to use as a reference point to enable him to determine his direction of motion. After the subject was suddenly halted to a stop, he slow-tracked in the clockwise direction he was previously traveling and fast reset in the opposite counterclockwise direction, but his eye movements jittered back and forth in the vertical and horizontal directions before that. This is because the subject was rotating so fast that the chair was shaking and slightly leaning so he bounced back and forth momentarily until he came to a halt. This will be further elaborated on, but the three semicircular canals may have been altered from their orthogonally positioned x, y and z planes. Inertia attempted to continue to move the endolymph in its direction of motion, but the endolymph and hair cells may have bounced back and forth causing the subject’s eyes to dart back and forth (and up and down) at the very beginning of the stop. Soon after, nystagmus (defined by direction of fast eye movement) was seen in direction of perceived motion or in the opposite counterclockwise direction because the abrupt stop caused the stereocilia to bend in the opposite direction.
In the final case, the procedures were the exact same as case 3, but the subject’s head was tilted over his left shoulder. After he was stopped and told to gaze forward with his head up, his eyes bounced up and down in the vertical direction moving slightly diagonally. This may be due to the fact that his tilted head misaligned the three orthogonally positioned planes in the semicircular canals. The vertical y-axis was tilted down into the plane of the x-axis so the endolymph fluid from the y-axis canal pooled into the x-axis canal. After the subject was stopped and his head was tilted upright, the patient perceived vertical acceleration. Based on the diagonal eye movements, it can be inferred that the z-axis may have also been misaligned and moved into the x-plane. The eyes were bouncing up and down rapidly because the nerves of the vestibulo-ocular reflex were still firing and the endolymph fluid had not completely reset into equilibrium in the three semicircular canals. This also led to a postural change as well because the subject felt as if he was falling backwards so he leaned forward in a misguided effort to compensate for what he perceived to be a lack of balance (St George et al., 2010, p. 2).
In conclusion, reflexes are essential in helping us to adapt to our environment and stronger stimuli should naturally generate a stronger and quicker response as demonstrated by the shorter latency period of the rapidly generated M-wave. Recurrent inhibition is another ingenious physiological defense mechanism that prevents fatigue and overstimulation via a negative feedback loop. Likewise with the complex polysynaptic vestibulo-ocular reflex arc vs. the monosynaptic stretch reflex, the vestibulo-ocular reflex can integrate so much more stimuli due to the presence of interneurons and afferents that can synapse onto multiple efferent motor neurons to affect multiple processes in the body.
Works Cited Page
Bautista, E and Korber, J. NPB 101L Systemic Physiology Lab Manual. 2nd ed. Mason, OH: Cengage Learning, 2008.
Misiaszek, JE. The H-reflex as a Tool in Neurophysiology: Its Limitations and Uses in Understanding Nervous System Function. Muscle Nerve. 2003; 28: 144-160.
Nishimaru, H, Koganezawa, T, Kakizaki, M, Ebihara, T, and Yanagawa, Y. Inhibitory Synaptic Modulation of Renshaw Cell Activity in the Lumbar Spinal Cord of Neonata Mice. Journal of Neurophysiology. 2010; 103: 3437′”3447.
Palmieri, R.M., C.D. Ingersoll, M.A. Hoffman, 2004. The Hoffman Reflex: Methodologic Considerations and Applications for Use in Sports Medicine and Athletic Training Research. Journal of Athletic Training. 2004; 39 (3): 268-277.
Sherwood, Lauralee. Human Physiology From Cells to Systems. 7th ed. Belmont, CA: Brooks/Cole, 2007.
St George, RJ, Day, BL, and Fitzpatrick, RC. Adaptation of Vestibular Signals for Self-Motion Perception. The Journal of Physiology. 2010; 1-21.