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🧪 Physiology Laboratory Skills — Complete Viva Guide (Q&A + Cross Questions)

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1. Preparation of a Neuromuscular Preparation

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2. Galvani's First Experiment

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3. Galvani's Second Experiment

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4. Matteucci Experiment — Secondary Tetanus

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5. Summation of Stimuli

a) Successive (Temporal) Summation

b) Spatial Summation

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6. Determination of Reflex Time (Türk's Experiment)

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7. Dynamometry

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8. Study of the Patellar Tendon Reflex (Knee Jerk)

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9. Brachial Extensor Reflex (Triceps Reflex)

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10. Definition of the Achilles Reflex (Ankle Jerk)

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11. Statokinetic Reflexes

• Static (postural) reflexes: maintain posture against gravity — tonic labyrinthine reflexes, tonic neck reflexes
• Statokinetic (kinetic) reflexes: maintain balance during movement — righting reflexes, Bárány's rotational reflexes, nystagmus, optokinetic reflex

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12. Danini–Aschner Reflex (Oculocardiac Reflex)

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13. Effect of Adrenaline on the Pupil of a Frog's Eye

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14. Esthesiometry (Tactile Discrimination)

• Fingertip: 2–5 mm (highest density of Meissner's corpuscles)
• Palm: 10–15 mm
• Forearm: 30–40 mm
• Back: 40–70 mm

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15. Determination of Visual Acuity

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16. Definition of the Field of View (Visual Field)

• Optic nerve lesion → monocular blindness
• Optic chiasm (central) → bitemporal hemianopia
• Optic tract → contralateral homonymous hemianopia
• Optic radiation (Meyer's loop) → contralateral superior quadrantanopia ("pie in the sky")
• Complete parietal optic radiation → contralateral homonymous hemianopia with macular sparing (PCA supply)

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17. Definition of Color Perception

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18. Determination of Hearing Acuity

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19. Comparison of Bone and Air Conduction

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20. Development of Conditioned Reflexes in Humans

• Classical (Pavlovian): Conditioned stimulus (CS) paired with unconditioned stimulus (UCS) → conditioned response (CR). Reflexive, involuntary. Example: salivation to a bell.
• Operant (Skinnerian): Behavior followed by reinforcement → increased frequency of behavior. Voluntary, learned.

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📋 Quick Summary Table — Key Facts for Viva

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Textbook basis: Ganong's Review of Medical Physiology, 26th Edition — sections on stretch reflexes, conditioned learning, and the knee jerk (Clinical Box 12-1), confirming the monosynaptic arc of the patellar reflex, grading system, and clinical interpretation; and Neuroanatomy through Clinical Cases, 3rd Edition — Table 3.6 on deep tendon reflexes and root levels.

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🧪 PHYSIOLOGY LABORATORY SKILLS — COMPLETE VIVA GUIDE

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LAB 1: PREPARATION OF A NEUROMUSCULAR PREPARATION

What It Is

• A classical experimental model in physiology
• Consists of the sciatic nerve + gastrocnemius muscle of a frog, kept intact together
• Used to study excitability, conduction, contraction, fatigue, and pharmacology of nerve-muscle

Why Frog Is Used

• Lower metabolic rate → tissues survive longer outside the body
• Simple, cheap, easy dissection
• Responds well to electrical stimulation
• Frog Ringer's solution keeps tissue viable for hours

Steps of Preparation

1. Destroy the frog's brain and spinal cord by pithing (insert a needle through foramen magnum)
2. Pin the frog dorsal side up on a dissection board
3. Remove skin from lower limbs
4. Isolate the sciatic nerve carefully from the thigh muscles
5. Cut the femur at mid-shaft, leaving knee and below intact
6. Separate gastrocnemius muscle from the leg, keeping Achilles tendon long
7. Tie a thread to the Achilles tendon for attachment to myograph
8. Place preparation in a moist chamber with frog Ringer's solution

Key Precautions

• Never touch the nerve with metal instruments (use glass hooks)
• Keep moist with Ringer's solution throughout
• Avoid stretching or traumatizing the nerve
• Handle muscle by tendon only, not the belly
• Work quickly to minimize drying

Frog Ringer's Solution Composition

• Normal saline alone is NOT adequate — lacks K⁺ and Ca²⁺ needed for excitation-contraction coupling

Viva Q&A

• Lacks K⁺ → membrane potential disturbed
• Lacks Ca²⁺ → no neuromuscular transmission, no muscle contraction

• Minimum current of infinite duration required to produce a threshold response

• Duration of stimulus at twice rheobase that produces threshold response
• Measure of tissue excitability — shorter chronaxie = more excitable
• Nerve fibers: ~0.01–0.1 ms; muscle fibers: ~1–10 ms

• Na⁺/K⁺-ATPase pump (electrogenic, pumps 3 Na⁺ out, 2 K⁺ in)
• High K⁺ permeability at rest (K⁺ leak channels)
• RMP of frog nerve ≈ –70 mV

• Failure of synaptic vesicle fusion at NMJ → no ACh release → no muscle contraction
• Also affects muscle contraction directly (no Ca²⁺ release from SR via CICR)

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LAB 2: GALVANI'S FIRST EXPERIMENT

Background

• Luigi Galvani (1737–1798), Italian physician
• Discovered that electrical stimulation caused dead frog muscle to twitch
• Believed electricity was intrinsic to animal tissue = "animal electricity"

Experimental Setup

• Neuromuscular preparation placed on a metal sheet (e.g., zinc)
• The sciatic nerve is touched with a different metal (e.g., copper/silver) rod
• The two metals are connected externally → completes a circuit → muscle twitches

What Actually Happens

• Two dissimilar metals create a bimetallic junction (electrochemical potential difference)
• This current flows through the tissue as the living tissue acts as an electrolyte
• Muscle contracts — acting as a biological galvanoscope (current detector)

Conclusion Galvani Drew (Incorrect)

• Electricity originated FROM the animal tissue ("animal electricity")

True Explanation (Volta's Correction)

• Electricity arose from the contact of two dissimilar metals (bimetallic cell)
• This led Volta to invent the voltaic pile (first electric battery, 1800)
• The tissue acted as an electrolyte conductor, NOT as a source

Viva Q&A

• Acts as a biological galvanoscope — detects the passage of electric current by contracting

• Electricity came from the dissimilar metals, not from the animal
• Proved by using bimetallic strips without any animal tissue — still generated current

• Typically zinc and copper, or iron and silver — any two dissimilar metals with different electrode potentials

• Contact of two dissimilar metals in an electrolyte creates a potential difference due to different oxidation potentials → generates current

• YES, partially — biological tissues do generate bioelectric potentials (action potentials, injury currents)
• Galvani's SECOND experiment proved this more correctly
• His first experiment error was attributing bimetallic electricity to the animal

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LAB 3: GALVANI'S SECOND EXPERIMENT

Setup

• One neuromuscular preparation (Prep 1) is laid flat
• The sciatic nerve of Prep 1 is folded over and laid on the cut (injured) surface of its own muscle
• OR: the nerve of a second preparation is placed across the muscle of Prep 1 at a cut surface

What Happens

• The muscle of Prep 1 contracts rhythmically
• This is the current of injury (demarcation potential) stimulating the nerve

Current of Injury (Demarcation Potential)

• When a muscle/nerve is cut → injured surface depolarizes (becomes electrically negative)
• Intact surface remains at resting potential (negative inside ≈ –70 mV)
• This creates a potential difference between cut end (–) and intact surface (+)
• This small current (~30–60 mV) is sufficient to stimulate the nearby nerve

Significance

• First evidence of endogenous bioelectric potentials in living tissue
• Foundation of modern electrophysiology (ECG, EEG, EMG, nerve conduction studies)
• No external electricity was used — tissue's own electricity caused the effect

Viva Q&A

• Potential difference between injured (depolarized, electronegative) and intact surface of muscle/nerve
• Magnitude: ~30–60 mV

• ~–70 mV in nerve, ~–90 mV in muscle
• Due to: K⁺ leak channels (K⁺ flows out) + Na⁺/K⁺ pump + impermeant intracellular anions

• EMG (electromyography) — records electrical potentials from muscles
• Nerve Conduction Studies (NCS) — records action potentials in nerves
• ECG — records cardiac bioelectric potentials
• EEG — records brain electrical activity

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LAB 4: MATTEUCCI EXPERIMENT — SECONDARY TETANUS

Setup

• Preparation 1: Nerve-muscle prep placed on electromagnetic stimulator → stimulated to produce tetanus (primary tetanus)
• Preparation 2: Its sciatic nerve is laid across the contracting muscle of Preparation 1

What Happens

• The contracting muscle of Prep 1 generates rhythmic action currents
• These electrical impulses spread to the nerve of Prep 2
• Prep 2 also shows tetanic contraction → called Secondary Tetanus

What This Proves

• Contracting muscles generate measurable electrical activity (action potentials)
• These muscle action currents are strong enough to stimulate an adjacent nerve
• Predecessor to modern EMG concept

Tetanus — Definition and Types

Mechanism of Tetanus

• Rapid stimuli → Ca²⁺ released from SR faster than it is resequestered
• Ca²⁺ remains elevated → troponin C remains bound → cross-bridges remain active
• Sustained contraction (4–5× force of single twitch)

Viva Q&A

• Primary = tetanus of Prep 1 caused by direct electrical stimulation
• Secondary = tetanus of Prep 2 caused by the electrical currents from Prep 1's contracting muscle — NO direct stimulation of Prep 2

• Physiological tetanus = sustained fused muscle contraction from high-frequency stimulation
• Tetanus disease = caused by Clostridium tetani toxin (tetanospasmin) blocking glycine/GABA inhibitory interneurons → spastic paralysis

• Electromyography (EMG) — needle or surface electrodes record electrical activity of muscle during contraction

• At low frequencies, twitches add incompletely (incomplete tetanus)
• At high frequencies, no relaxation between stimuli → complete fusion = complete tetanus
• Mechanism: residual Ca²⁺ accumulates with each stimulus

• To increase muscle force: either increase firing rate of active motor units (rate coding) OR recruit additional motor units (size principle — small → large)

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LAB 5: SUMMATION OF STIMULI

a) Successive (Temporal) Summation

Definition

• Two subthreshold stimuli applied to the same point in rapid succession
• If the second arrives before the first local potential has fully decayed → the two depolarizations add together → reach threshold → action potential

Mechanism

• First stimulus: creates a local graded potential (does NOT reach threshold alone)
• This potential decays over time but not instantly
• Second stimulus: arrives while membrane is still partially depolarized
• Combined effect crosses threshold → action potential fires

Conditions Required

• Interval between stimuli must be > absolute refractory period (>~1 ms)
• Interval must be < time for full recovery of membrane potential
• Each individual stimulus must be subthreshold

Key Points

• The action potential follows all-or-none law — either fires fully or not at all
• Summation is at the local potential level, not the action potential level
• Occurs at both nerve fiber level and synapse level (temporal summation of EPSPs)

b) Spatial Summation

Definition

• Two subthreshold stimuli applied to different points simultaneously
• Their effects converge on the same excitable membrane
• Combined depolarization reaches threshold → action potential

Mechanism

• Different receptor sites/synaptic inputs simultaneously depolarize the membrane
• At neurons: multiple EPSPs from different presynaptic axons arrive simultaneously at the same postsynaptic neuron → summate → threshold reached

Most Important Site

• Motor neuron soma/dendrites in the anterior horn
• Receives convergent input from thousands of presynaptic neurons
• Integration of all EPSPs and IPSPs determines whether motor neuron fires

Viva Q&A

• Excitatory postsynaptic potential — local depolarization of postsynaptic membrane
• Caused by: opening of Na⁺ (and K⁺) channels via ionotropic receptors (e.g., AMPA, NMDA)
• Brings membrane closer to threshold (~–55 mV)

• Inhibitory postsynaptic potential — hyperpolarization of postsynaptic membrane
• Caused by: opening of Cl⁻ channels (GABA-A, glycine receptors) or K⁺ channels (GABA-B)
• Moves membrane away from threshold

• NO — action potentials are all-or-none; once threshold is reached, a full AP fires
• Only local/graded potentials summate

• Period during which NO stimulus, however strong, can fire another AP
• Caused by: Na⁺ channels in inactivated state (h-gate closed)
• Duration: ~1–2 ms (limits max firing rate)

• Follows absolute refractory period
• A suprathreshold stimulus CAN fire an AP but it requires stronger stimulus
• Caused by: continued K⁺ efflux (after-hyperpolarization) + partial Na⁺ channel recovery

• Myelinated fibers conduct faster → APs arrive more rapidly → more effective temporal summation per unit time
• Also, saltatory conduction reduces the distance over which local currents decay

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LAB 6: DETERMINATION OF REFLEX TIME (TÜRK'S EXPERIMENT)

Definition of Reflex Time

• Total time from application of stimulus to onset of reflex response
• Also called reflex latency

Components of Reflex Time

1. Receptor activation time — stimulus → receptor potential
2. Afferent conduction time — receptor → spinal cord (depends on nerve length + velocity)
3. Central (synaptic) delay — 0.5 ms per synapse; dominant variable for polysynaptic reflexes
4. Efferent conduction time — spinal cord → effector organ
5. Effector latency — NMJ delay + muscle contraction initiation (~1–3 ms)

Türk's Experiment Procedure

1. Frog is pithed in the brain only (spinal cord intact)
2. Suspended vertically by its jaw
3. Hind limb dipped into dilute H₂SO₄ solution (acid stimulates skin nociceptors)
4. Time from dipping to leg withdrawal is measured with a stopwatch
5. Experiment is repeated with different concentrations (stimulus intensities) and different dip depths (different levels of limb)

Results and Analysis

• Stronger stimulus (higher acid concentration) → shorter reflex time
• Deeper dip (more nociceptors stimulated) → shorter reflex time (spatial summation)
• Difference in reflex times between different levels allows calculation of conduction velocity
• By subtracting peripheral conduction times, central delay can be estimated

Why Stronger Stimulus → Shorter Reflex Time

• More afferent fibers recruited simultaneously (spatial summation)
• Larger/faster EPSP generation at motor neuron
• Threshold reached sooner → faster firing of efferent neuron

Viva Q&A

• ~0.5 ms per synapse
• Time for: Ca²⁺ influx into presynaptic terminal → vesicle fusion → ACh/neurotransmitter diffusion across cleft → receptor binding → EPSP generation

• One (monosynaptic) — Ia afferent → α-motor neuron
• Therefore has the shortest possible central delay (~0.5 ms)

• Flexor withdrawal reflex (polysynaptic nociceptive reflex)
• Multiple synapses → longer, measurable central delay

• Patient clasps hands and pulls them apart (isometric contraction of arm muscles) while knee jerk is tested
• Enhances the patellar reflex
• Mechanism: increased γ-motor neuron activity → increased muscle spindle sensitivity → lower threshold for reflex firing

• Theoretically only for a monosynaptic reflex with zero distance (impossible physically)
• In practice, minimum reflex time limited by: receptor latency + 1 synaptic delay + minimal conduction + NMJ + contraction latency ≈ ~20–30 ms even for patellar reflex

• Cold slows enzyme kinetics, ion channel kinetics, and nerve conduction → increases reflex time
• Warm speeds up all above → decreases reflex time

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LAB 7: DYNAMOMETRY

Definition

• Measurement of muscle strength using a dynamometer
• Most commonly measures hand grip strength

Types of Dynamometers

Procedure (Hand Grip)

1. Patient stands or sits with elbow at 90°, forearm neutral
2. Handgrip dynamometer placed in dominant hand
3. Squeeze as hard as possible for 3–5 seconds
4. Repeat 3 times; record best or average
5. Compare with normative values (adjusted for age, sex, hand dominance)

Normal Values

• Dominant hand typically 10% stronger than non-dominant

Factors Affecting Results

• Intrinsic: Age (peak at 30–40 years), sex (males > females), training status, nutrition
• Extrinsic: Time of day (AM slightly lower), motivation, pain, fatigue, technique
• Pathological: Myopathy, neuropathy, tendinopathy, arthritis, stroke (contralateral weakness)

Clinical Significance

• Low grip strength = predictor of sarcopenia, frailty, mortality in elderly
• Used in nutritional assessment (protein-energy malnutrition)
• Monitored in rehabilitation after stroke, orthopedic surgery
• Occupation-specific injury assessment

Types of Muscle Contraction

Viva Q&A

• Progressive decline in force production despite continued stimulation
• Mechanisms:
  • Accumulation of H⁺, Pi (inorganic phosphate), ADP → impairs cross-bridge cycling
  • Impaired Ca²⁺ release from sarcoplasmic reticulum
  • Depletion of glycogen and ATP
  • Central fatigue: reduced motor drive from CNS

• Inverse relationship: greater load (resistance) → slower contraction velocity
• At zero load → maximum velocity (Vmax)
• At maximum load (isometric) → zero velocity but maximum force

• Optimal sarcomere length (~2.2 μm in humans) → maximum cross-bridge overlap → maximum force
• Too short: thick filaments hit Z-lines, reduced overlap → less force
• Too long: reduced overlap of thin and thick filaments → less force

1. Powering myosin head cross-bridge detachment (ATPase)
2. Ca²⁺ reuptake into SR (SERCA pump)
3. Na⁺/K⁺ pump to restore membrane potential
4. Actomyosin rigor state relief (rigor mortis when ATP depleted)

• After death, ATP is depleted → myosin heads cannot detach from actin → permanent actomyosin binding → rigidity
• Begins 2–6 hours after death, maximal at 12 hours, resolves after 24–48 hours (proteolysis)

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LAB 8: STUDY OF THE PATELLAR TENDON REFLEX

Definition

• A monosynaptic stretch reflex (deep tendon reflex / DTR)
• Also called: Knee jerk reflex or quadriceps reflex

Reflex Arc (Point by Point)

1. Stimulus: Tap on patellar tendon → sudden stretch of quadriceps femoris muscle
2. Receptor: Muscle spindle (Ia afferent endings on nuclear bag/chain fibers) detects stretch
3. Afferent: Ia (Group Ia) fibers — fastest conducting (70–120 m/s), heavily myelinated, largest diameter
4. Center: Enters spinal cord at L2–L4 → dorsal horn → synapses DIRECTLY on α-motor neuron in anterior horn (ONE synapse)
5. Efferent: α-motor neuron → motor fibers in femoral nerve
6. Effector: Quadriceps femoris contracts → knee extension
7. Simultaneous: Ia afferent also → Ia inhibitory interneuron → inhibits hamstring α-motor neurons (reciprocal inhibition)

Grading Scale (NIMS Grading)

Clinical Implications

Viva Q&A

• Only ONE synapse: between Ia afferent and α-motor neuron
• No interneurons in the excitatory pathway
• Therefore shortest possible reflex arc

• Encapsulated sensory organ embedded in muscle parallel to extrafusal fibers
• Contains: intrafusal fibers (nuclear bag + nuclear chain), Ia and II afferents, γ-motor neurons (fusimotor)
• Detects: muscle stretch (length changes)

• When α-motor neurons are activated to contract extrafusal fibers, γ-motor neurons also fire simultaneously
• This maintains muscle spindle tension during contraction → spindle doesn't go slack → can still detect changes in length

• When quadriceps contracts (patellar reflex), hamstrings are simultaneously inhibited
• Via: Ia afferent → Ia inhibitory interneuron → inhibits hamstring α-motor neurons
• Purpose: smooth, coordinated movement without antagonist resistance

• Rhythmic, involuntary muscle contractions at ~5–8 Hz in response to sustained muscle stretch
• Indicates severe UMN lesion with exaggerated stretch reflex
• Test: rapidly dorsiflex foot and hold → repeated plantar flexion oscillations

• Stroking plantar surface of foot → extension of big toe + fanning of others (positive Babinski)
• Indicates UMN lesion (normal in infants up to 18 months)
• Normal response = plantar flexion of all toes

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LAB 9: BRACHIAL EXTENSOR REFLEX (TRICEPS REFLEX)

Definition

• A monosynaptic stretch reflex of the triceps brachii muscle
• Tests the C7 nerve root and radial nerve integrity

Reflex Arc

1. Stimulus: Tap on triceps tendon (just above olecranon)
2. Receptor: Muscle spindle in triceps brachii
3. Afferent: Ia fibers → radial nerve → enters spinal cord at C7 (also C6, C8)
4. Center: Synapse on α-motor neurons in anterior horn C7
5. Efferent: α-motor neuron → radial nerve → triceps brachii
6. Response: Elbow extension

How to Elicit

• Patient's elbow flexed at ~90°, forearm supported (hanging loose)
• Examiner taps triceps tendon firmly with reflex hammer
• Normal response: brief visible elbow extension

Comparison of Upper Limb Reflexes

Viva Q&A

• C7 radiculopathy (C7 disc herniation — C6/C7 disc level)
• Radial nerve palsy (Saturday night palsy — wrist drop + absent triceps reflex)
• Brachial plexus injury (posterior cord)

• UMN lesion above C7 (stroke, MS, cervical myelopathy, ALS)

• Radial nerve compression at spiral groove of humerus (arm draped over chair during sleep/intoxication)
• Results in: wrist drop, finger drop, absent brachioradialis reflex, absent triceps reflex (if above spiral groove)
• Sensation lost over dorsum of hand and lateral forearm

• Radial nerve at axilla/spiral groove: wrist drop + finger drop + sensory loss + absent triceps reflex
• Posterior interosseous nerve (deep branch) below lateral epicondyle: finger drop (extensors) but NO wrist drop, NO sensory loss (pure motor branch)

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LAB 10: DEFINITION OF THE ACHILLES REFLEX

Definition

• A monosynaptic stretch reflex of gastrocnemius/soleus muscles
• Also called: Ankle jerk or ankle reflex

Reflex Arc

1. Stimulus: Tap on Achilles tendon
2. Receptor: Muscle spindle in gastrocnemius/soleus
3. Afferent: Ia fibers → tibial nerve → sciatic nerve → enters cord at S1 (also S2)
4. Center: Synapse on α-motor neurons at S1, S2 anterior horn
5. Efferent: Tibial nerve
6. Response: Plantar flexion of foot

How to Elicit

• Patient kneeling on a chair or lying prone with foot relaxed
• Slightly dorsiflex foot to prestretch the gastrocnemius
• Tap the Achilles tendon smartly
• Look for plantar flexion

Clinical Significance

Viva Q&A

• S1 afferents to gastrocnemius are the longest large-diameter fibers in the body
• Length-dependent neuropathies (DM, alcohol, Charcot-Marie-Tooth) affect longest fibers first
• Therefore ankle jerk lost before knee jerk

• Normal contraction phase but prolonged, slow relaxation
• Mechanism: slow Ca²⁺ reuptake by SERCA pump in hypothyroid state (thyroid hormones regulate SERCA expression)
• Also reduced ATP production → slower cross-bridge cycling

• Patient lies supine, examiner externally rotates and slightly abducts the hip
• Flex knee slightly, hold foot in slight dorsiflexion
• Tap Achilles tendon

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LAB 11: STATOKINETIC REFLEXES

Definition

• Reflexes that maintain posture, equilibrium, and orientation of the body in space during rest and movement
• Arise from: vestibular apparatus, visual system, proprioceptors, joint receptors

Classification

A. Static (Tonic/Postural) Reflexes

• Maintain posture against gravity without movement

1. Tonic labyrinthine reflexes — otolith organs detect head position → adjust limb and trunk muscle tone
2. Tonic neck reflexes — neck muscle proprioceptors detect head rotation/flexion → adjust limb tone
3. Tonic body righting reflexes — body asymmetric contact with surface → righting response

B. Phasic (Statokinetic) Reflexes

• Respond to movement and acceleration

1. Righting reflexes — restore normal body orientation after displacement
   • Labyrinthine righting (otolith → correct head position)
   • Optical righting (visual input → correct head/body)
   • Neck righting (neck → correct body orientation)
   • Body-on-body righting (body contact → correct position)
2. Otolith reflexes — compensate for linear acceleration (lift/tilt)
3. Rotational (vestibulo-ocular) reflex — semicircular canals detect angular acceleration → compensatory eye movement (VOR)
4. Nystagmus — rhythmic eye movement to maintain visual fixation during head rotation
5. Optokinetic reflex — visual motion field → compensatory eye tracking

Vestibular Apparatus Review

Integration Centers

• Vestibular nuclei (medulla/pons — Deiter's nucleus, Bechterew's nucleus)
• Cerebellum (flocculonodular lobe = vestibulocerebellum)
• Spinal cord via vestibulospinal tracts (lateral and medial)
• Brain stem (for VOR and nystagmus)

Viva Q&A

• Head rotates right → semicircular canal signals → eyes move LEFT to maintain visual fixation
• Allows clear vision during head movement
• Gain = 1 (eye movement exactly opposite to head movement)

• Rhythmic alternating eye movement: slow phase (toward stimulus) + fast corrective saccade (away)
• Named by direction of fast phase
• Physiological: rotation-induced, caloric-induced
• Pathological: at rest without stimulus → indicates vestibular/cerebellar/brainstem pathology

• Romberg test, Unterberger stepping test, head impulse test, caloric testing (COWS: Cold Opposite Warm Same), Dix–Hallpike test (for BPPV)

• Benign Paroxysmal Positional Vertigo
• Displaced otoconia (calcium carbonate crystals) from utricle → float into posterior semicircular canal
• Triggered by head position changes → brief vertigo + upbeat-torsional nystagmus
• Treated by Epley maneuver (canalith repositioning)

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LAB 12: DANINI–ASCHNER REFLEX (OCULOCARDIAC REFLEX)

Definition

• Application of pressure on closed eyeballs (or traction on extraocular muscles) → reflex bradycardia
• Normal response: heart rate decreases by 5–13 bpm (or 10–20%)

Reflex Arc

Mechanism of Bradycardia

• Vagal activation → ACh released → binds M₂ muscarinic receptors on SA node
• Activates IKACh (inward rectifier K⁺) channels → K⁺ efflux → hyperpolarization of SA node
• Pacemaker potential slope decreases → rate of spontaneous depolarization slows → bradycardia

How to Test (Clinical)

1. Measure baseline heart rate (pulse or ECG)
2. Apply gentle, sustained, equal bilateral pressure on closed eyelids for 20–30 seconds
3. Measure heart rate during and after
4. Drop of >10% = positive/exaggerated response

Clinical Importance

• Operative: Can cause severe bradycardia, arrhythmia, or asystole during:
  • Strabismus surgery (most common trigger)
  • Enucleation, retrobulbar block
  • Ocular trauma
• Prevention: Atropine preoperatively (IV or IM)
• Treatment: Stop surgical manipulation → atropine if persists → CPR if asystole

Viva Q&A

• Afferent: Trigeminal nerve (V₁ — ophthalmic branch)
• Efferent: Vagus nerve (CN X)

• Atropine (muscarinic antagonist)
• Blocks M₂ receptors on SA node → prevents vagal bradycardia

• ACh → M₂ receptor activation → Gi protein → ↑K⁺ conductance (IKACh) + ↓cAMP → slowed SA node spontaneous depolarization

• Severe vagal stimulation → high-degree AV block → ventricular bradycardia or asystole
• Mechanism: ACh shortens AV node refractory period AND slows conduction velocity

• Carotid sinus reflex (baroreceptor reflex)
• Bezold–Jarisch reflex (ventricular mechanoreceptors → vasovagal syncope)
• Diving reflex (face in cold water → bradycardia via trigeminal V afferent → vagal efferent — same pathway as Aschner!)
• Valsalva response (phase II)

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LAB 13: EFFECT OF ADRENALINE ON THE PUPIL OF A FROG'S EYE

Expected Effect

• Mydriasis (pupil dilation) — adrenaline causes the pupil to enlarge

Iris Muscles and Their Innervation

Mechanism of Adrenaline-Induced Mydriasis

1. Adrenaline (epinephrine) binds α₁-adrenoceptors on dilator pupillae
2. Gq-protein → IP₃/DAG → ↑intracellular Ca²⁺ → smooth muscle contraction
3. Dilator muscle contraction → iris pulled radially toward periphery → pupil widens

Why Frog Is Used

• Frog iris is large and visible
• Thin, transparent cornea allows direct observation
• Good response to topically applied drugs

How to Do the Experiment

1. Sacrifice frog, remove one eye, place in Ringer's solution
2. Apply 1–2 drops of 1:1000 adrenaline solution to the corneal surface
3. Observe pupil size every 2–5 minutes (compare with untreated eye as control)
4. Measure pupil diameter under a magnifying glass/ruler

Viva Q&A

1. First-order neuron: Hypothalamus → ciliospinal center of Budge (C8–T2)
2. Second-order (preganglionic): T1 → over apex of lung → superior cervical ganglion (SCG)
3. Third-order (postganglionic): SCG → along internal carotid artery → via ophthalmic branch → dilator pupillae

• Disruption of sympathetic pathway at ANY of the three levels
• Triad:
  • Miosis (dilator paralysis)
  • Partial ptosis (paralysis of Müller's superior tarsal muscle)
  • Anhidrosis (ipsilateral face, depending on lesion level)
• ± Enophthalmos (apparent, not real)

• Selective α₁ agonist → mydriasis (same as adrenaline but no beta effects)
• Used clinically for pupil dilation without cycloplegia

• Pilocarpine (M agonist — contracts sphincter + ciliary muscle)
• Opioids (morphine, heroin — "pinpoint pupils" via central parasympathetic activation)
• Organophosphates (acetylcholinesterase inhibitors → excess ACh → miosis)

• Light → retina → optic nerve → optic chiasm → pretectal nuclei (midbrain) → Edinger-Westphal nucleus → CN III → ciliary ganglion → sphincter pupillae → miosis
• Direct reflex: same eye
• Consensual reflex: opposite eye constricts too
• Tests: CN II (afferent), CN III (efferent), pretectal area integrity

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LAB 14: ESTHESIOMETRY (TACTILE SPATIAL DISCRIMINATION)

Definition

• Esthesiometry = measurement of tactile sensation threshold and spatial discrimination
• Most common test: two-point discrimination (2PD) — minimum distance at which two simultaneous touch stimuli are perceived as two separate points

Instrument

• Esthesiometer / Weber Compass / Calibrated calipers / Aesthesiometer
• Electronic von Frey aesthesiometer (modern)

Procedure

1. Patient sits with eyes closed
2. Two-point compass is applied to the skin simultaneously with both tips
3. Start with large separation (e.g., 5 cm) and decrease until patient reports feeling only ONE point
4. The minimum distance at which TWO points are felt = two-point discrimination threshold
5. Test multiple body regions; compare bilaterally

Normal Values (Two-Point Discrimination)

Why Fingertip Has Best Discrimination

• Highest density of Meissner's corpuscles
• Smallest receptive fields
• Greatest cortical representation (homunculus — fingers and lips are disproportionately large)

Sensory Receptors for Fine Touch

Viva Q&A

1. Receptor density (number of receptors per unit area)
2. Receptive field size (smaller = better discrimination)
3. Cortical magnification factor (cortical area devoted to that body part)
4. Neural processing in dorsal column–medial lemniscus pathway

• Dorsal column–medial lemniscus pathway
• Receptor → Aβ fibers → dorsal column (nucleus gracilis/cuneatus) → decussate in medulla → medial lemniscus → thalamus (VPL) → primary somatosensory cortex (S1, postcentral gyrus)

• Primary somatosensory cortex (S1) — postcentral gyrus, Brodmann areas 3a, 3b, 1, 2
• Organized as a somatotopic map (homunculus)
• Secondary somatosensory cortex (S2) — superior temporal/parietal region

• Ipsilateral: loss of fine touch, proprioception, vibration (dorsal column)
• Contralateral: loss of pain and temperature (spinothalamic tract — crosses 1–2 levels above entry)
• Ipsilateral: UMN signs (corticospinal tract) below lesion

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LAB 15: DETERMINATION OF VISUAL ACUITY

Definition

• Visual acuity = the ability to resolve two closely spaced points as distinct
• Expressed as the minimum angle of resolution (MAR) — normal = 1 minute of arc

Snellen Chart Method

• Standard test at 6 meters (20 feet)
• Each letter designed so its strokes subtend 1 minute of arc at the designated distance
• Largest letter (top) readable at 60 m; smallest (bottom) at 5 m

Notation

• 6/6 (metric) = 20/20 (imperial) = NORMAL
• 6/12: reads at 6 m what normal reads at 12 m → visual acuity halved
• 6/60: reads at 6 m what normal reads at 60 m → severely reduced

Testing Procedure

1. Test each eye separately (cover the other with an opaque occluder)
2. Patient stands 6 m from chart in good illumination
3. Ask to read smallest line possible
4. Record the line number (distance denominator)
5. If less than 6/60: test at 3 m (3/60), count fingers (CF), hand movements (HM), perception of light (PL), no perception of light (NPL)

Factors Affecting Visual Acuity

Anatomical Basis of Maximum Acuity

• Fovea centralis (central macula)
• Contains only cones — ~150,000–200,000 cones/mm²
• 1:1 ratio — each cone connects to one bipolar → one ganglion cell → private line
• No rod interference, no convergence
• Receives maximum blood supply (central artery of retina)

Viva Q&A

• Perfectly refracted eye — parallel rays from infinity focused exactly on retina without accommodation
• Focal length matches axial length

• Eyeball too long (or cornea too curved) → parallel rays focused ANTERIOR to retina
• Corrected with concave (–) lens

• Eyeball too short → parallel rays focused POSTERIOR to retina
• Corrected with convex (+) lens

• Unequal curvature of cornea in different meridians → different focal points → blurred image
• Corrected with cylindrical lenses

• Age-related loss of accommodation (near vision) due to loss of lens elasticity and ciliary muscle power
• Usually begins after age 40
• Corrected with reading glasses (+) / bifocals

• Ring with a gap; patient identifies gap orientation
• Eliminates letter recognition bias
• Used for illiterates, children, cross-cultural testing

• Fovea: temporal to optic disc (temporal macula), slightly inferior
• Optic disc: nasal, approximately 15° from fovea
• On fundoscopy: optic disc on nasal side, macula/fovea on temporal side with central foveal reflex

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LAB 16: DEFINITION OF THE FIELD OF VIEW (VISUAL FIELD)

Definition

• Visual field: total area visible to one eye while looking straight ahead without eye movement
• Normal monocular field: ~60° nasal, 90° temporal, 60° superior, 75° inferior
• Binocular field: ~200° (with ~120° overlap zone for stereoscopic vision)

How to Measure

Confrontation Test (Bedside)

• Patient covers one eye, stares at examiner's nose
• Examiner holds fingers equidistant between them
• Patient reports when fingers become visible from periphery
• Compare patient's field to examiner's (assumes examiner has normal field)

Perimetry (Formal)

• Goldmann perimetry (kinetic): moving target from periphery to center; maps isopters
• Automated perimetry (Humphrey field analyzer): static test; measures threshold sensitivity at each point; gold standard

The Blind Spot

• ~15° temporal from fixation point in each eye
• Corresponds to the optic nerve head (optic disc) — no photoreceptors
• Can be mapped by moving a small target until it disappears then reappears
• Size: ~5° × 7°

Visual Field Pathway and Lesion Mapping

Why Macular Sparing in Occipital Cortex Lesions?

• Macula has dual blood supply (MCA + PCA) — large cortical representation
• In MCA infarction, PCA territory (macular representation at occipital pole) is spared

Viva Q&A

• Nasal side of eye overlaps with nose → physical obstruction
• No physical obstruction on temporal side

• Nasal fibers (representing temporal visual field) cross to contralateral optic tract
• Temporal fibers (representing nasal visual field) remain ipsilateral
• Therefore: each optic tract carries information from the contralateral visual field from BOTH eyes

• Compresses optic chiasm from below (crossing nasal fibers)
• → Bitemporal hemianopia
• Classic presentation: patient bumps into door frames on both sides

• Severe concentric visual field constriction → only central vision remains
• Causes: advanced glaucoma (peripheral loss first), retinitis pigmentosa, bilateral occipital infarcts (sparing only macular fibers)

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LAB 17: DEFINITION OF COLOR PERCEPTION

Trichromatic Theory (Young–Helmholtz)

• Three types of cones: S (short/blue, 420 nm), M (medium/green, 530 nm), L (long/red, 560 nm)
• All colors perceived by differential stimulation of these three cone types
• Brain integrates relative stimulation of three cones → perceived color

Opponent-Color Theory (Hering)

• Three opponent channels at ganglion cell / LGN level:
  1. Red vs. Green
  2. Blue vs. Yellow
  3. Black vs. White (luminance)
• Explains: afterimages, why we don't see "reddish-green" (opponent suppression)
• Modern view: Both theories are correct at different levels — trichromatic at cone level, opponent at processing level

Types of Color Blindness

Genetics

• Red and green cone genes: OPN1LW and OPN1MW — both on X chromosome → X-linked recessive inheritance
• Blue cone gene: OPN1SW — chromosome 7
• Therefore: red-green color blindness ~8% of males, ~0.5% females

Tests for Color Vision

Viva Q&A

• Deuteranomaly (anomalous green cones) — affects ~5% of males
• Red-green color blindness overall: ~8% males, ~0.5% females

• They cannot distinguish the target color (e.g., red/orange) from the background (e.g., green)
• Because their M and L cones have overlapping/absent spectral sensitivity

• The ability to perceive the same color regardless of illumination spectrum
• Mechanism: cerebral cortex (V4) compares object color to surrounding illumination and corrects

• V4 (visual area 4) in the ventral visual stream (fusiform gyrus area)
• Lesion → cerebral achromatopsia (inability to perceive color despite intact cones)

• A color appears different depending on the surrounding colors
• Explained by lateral inhibition in color-opponent channels

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LAB 18: DETERMINATION OF HEARING ACUITY

Audiometry

• Pure-tone audiogram: plots hearing threshold (minimum audible level) vs. frequency
• Frequencies tested: 250, 500, 1000, 2000, 4000, 8000 Hz
• Threshold expressed in dB HL (Hearing Level) — dB above normal average threshold
• Normal: threshold <25 dB HL across all frequencies

Degree of Hearing Loss

Human Hearing Range

• Frequency: 20 Hz – 20,000 Hz
• Most sensitive range: 1000–4000 Hz (speech frequencies: 500–3000 Hz)
• Maximum sensitivity: ~3000–4000 Hz (related to ear canal resonance)

Audiogram Patterns

Bedside Tests

Viva Q&A

• Logarithmic ratio of sound pressure level relative to a reference
• 0 dB = threshold of normal hearing (~20 μPa)
• 10 dB = 10× power increase; 20 dB = 100×; 60 dB = normal conversation; 120 dB = pain threshold

• Age-related sensorineural hearing loss
• Progressive high-frequency loss starting ~40 years
• Mechanism: cumulative degeneration of outer hair cells at cochlear base (high-frequency region)
• +/– strial atrophy, spiral ganglion degeneration

• Caused by excessive sound exposure → damages outer hair cells
• Characteristic: 4000 Hz notch on audiogram (most sensitive to noise damage)
• Can be temporary (temporary threshold shift, TTS) or permanent (permanent threshold shift, PTS)

• Outer hair cells (OHCs): electromotility (prestin protein) → cochlear amplification
• When OHCs are damaged → reduced amplification → elevated threshold → hearing loss

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LAB 19: COMPARISON OF BONE AND AIR CONDUCTION

Key Tests

Rinne Test

• Instrument: 512 Hz tuning fork (best for speech frequencies; 128 Hz only tests low freq)
• Procedure:
  1. Strike tuning fork → place on mastoid process behind ear (bone conduction)
  2. When patient says they can no longer hear it → immediately move to external auditory meatus (air conduction)
  3. Ask: "Can you still hear it?"

Weber Test

• Procedure: Vibrating 512 Hz tuning fork placed on midline skull (forehead or vertex)
• Ask: "Where do you hear the sound?"

Why Weber Lateralizes to the Conductive-Loss Side

• Ambient background noise is reduced in the affected ear (blocked EAC/middle ear)
• This unmasking effect (occlusion effect) makes bone-conducted sound perceived louder on that side

Combined Rinne + Weber Interpretation

Air Conduction Pathway

Bone Conduction Pathway

Viva Q&A

• 256 Hz: vibration often felt as touch sensation → confuses patient (they feel it rather than hear it)
• 512 Hz: predominantly heard, minimal vibrotactile sensation, in speech frequency range

• Compare patient's bone conduction duration with examiner's (assumed normal)
• Reduced ABC in patient = sensorineural loss

• Outer ear: impacted wax, foreign body, otitis externa, atresia
• Middle ear: otitis media with effusion (glue ear), otosclerosis, ossicular discontinuity, tympanic membrane perforation, cholesteatoma

• Abnormal bone remodeling → fixation of stapes footplate to oval window
• Results in: progressive conductive hearing loss, Rinne negative, Weber lateralizes to affected ear
• Treatment: stapedectomy (replace stapes with prosthesis)

• Compare patient's bone conduction duration with examiner's while patient's EAC is occluded
• If BC improves with occlusion (occlusion effect) → cochlea is intact

• Bone conduction tuning fork on mastoid → occlude EAC → normal person and SNHL patient will hear LOUDER (occlusion effect)
• Conductive HL patient: no change (already occluded)

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LAB 20: DEVELOPMENT OF CONDITIONED REFLEXES IN HUMANS

Pavlov's Classical Conditioning

• Ivan Pavlov (1849–1936) — Nobel Prize 1904 (physiology of digestion)
• Discovered conditioned reflexes while studying salivary secretion in dogs

Key Terminology

Procedure to Establish Conditioned Reflex in Humans

1. Select a measurable UCS/UCR pair — e.g., bright light → pupillary constriction; or knee tap → knee jerk
2. Select a neutral CS — e.g., auditory tone, bell, or flashing light (different from UCS)
3. Paired trials: Present CS ~0.5 seconds BEFORE UCS, repeatedly (10–20 trials minimum)
4. Test trials: Present CS alone (without UCS) → observe if UCR now occurs to CS alone = CR formed
5. Measure: Latency, magnitude, frequency of CR

Requirements for Conditioning

1. CS must precede UCS (contiguity) — forward conditioning is optimal
2. Repeated pairings needed (10–100 depending on stimulus)
3. CS should be novel and neutral initially
4. Subject must be alert and attentive
5. UCS must be biologically significant (strong motivational value)

Types of Conditioning

Properties of Conditioned Reflexes

1. Extinction: Repeated CS alone (without UCS) → gradual weakening and disappearance of CR
2. Spontaneous recovery: After extinction, rest period → CR partially reappears spontaneously
3. Stimulus generalization: Similar stimuli to CS also elicit CR (e.g., bell at different pitch)
4. Stimulus discrimination: With training, respond ONLY to specific CS and not to similar stimuli
5. Higher-order conditioning: Established CS (CS₁) acts as UCS to condition new CS₂

Classical vs. Operant Conditioning

Neural Substrates

Viva Q&A

• Extinction = progressive loss of CR when CS presented repeatedly without UCS
• CR is NOT erased — it is actively inhibited by new inhibitory learning (CS → no UCS)
• Evidence: spontaneous recovery occurs after rest (inhibition fades, original association persists)

• After CS₁ (bell) is firmly conditioned → use CS₁ as the "UCS" to condition CS₂ (light)
• Eventually: light alone → CR (salivation), even though light was never paired with food
• Typically possible up to 2nd order in animals, 3rd–4th order possible in humans

• Systematic desensitization (treat phobias — pair relaxation with feared stimulus)
• Aversion therapy (pair alcohol with emetic drug — conditioned nausea to alcohol)
• Biofeedback (condition autonomic responses)
• Placebo effect (conditioned physiological response to inert stimulus)

• Lateral nucleus: receives CS (auditory cortex) and UCS (pain — via thalamus) inputs → site of synaptic plasticity / "fear memory storage"
• Central nucleus: outputs to hypothalamus (autonomic response), PAG (freezing), brainstem (HR, BP changes)
• Bilateral amygdala damage → Klüver–Bucy syndrome — inability to form fear conditioned responses, psychic blindness, hypersexuality, hyperorality

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📊 MASTER QUICK REFERENCE TABLE

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Sources: Ganong's Review of Medical Physiology, 26th Ed. — stretch reflexes (Ch. 12), conditioned learning (Ch. 15); Neuroanatomy through Clinical Cases, 3rd Ed. — DTR table (Table 3.6); Medical Physiology (Boron & Boulpaep) — NMJ physiology