The autonomic nervous system (ANS) is a very complex, multifaceted neural network that maintains internal physiologic homeostasis. This network includes cardiovascular, thermoregulatory, GI, genitourinary (GU), and ophthalmologic (pupillary) systems (see the following image). Given the complex nature of this system, a stepwise approach to autonomic disorders is required for proper understanding.
The goal for this article remains focused at step III on the anatomy of the autonomic nervous system, as follows.
Step I - Understand the reason for testing
Step II - Recognition and etiology (especially small fiber neuropathy [SFN])
Step III - Understand basic anatomy and neurophysiology
Step IV - Learn the methods for testing
Step V - Diagnosis and management
Almost 10% of the population (or > 30 million people in the US) may acquire an autonomic disorder requiring medical attention. Because the autonomic nervous system maintains internal physiologic homeostasis, disorders of this system can be present with both central as well as peripheral nervous system localization.
Causes of ANS dysfunction
The etiology of autonomic dysfunction can be primary or idiopathic and secondary causes. Autonomic failure is seen in multiple system atrophy, pure or progressive autonomic failure, Parkinson and other neurodegenerative diseases, metabolic diseases such as Wernicke and cobalamin deficiency, diabetes mellitus, hyperlipidemia, trauma, vascular diseases, neoplastic diseases, and multiple sclerosis. In addition, autonomic dysfunction is associated with various medications.
In addition to diabetes, autonomic dysfunction is associated with other neuropathies, including Guillain-Barré syndrome, Lyme disease, human immunodeficiency virus (HIV) infection, leprosy, acute idiopathic dysautonomia, amyloidosis, porphyria, uremia, and alcoholism. Besides nerve localization in the peripheral nervous system, it occurs in diseases of the presynaptic neuromuscular junction such as botulism and myasthenic syndrome.
In addition to the acquired causes, inherited disorders like hereditary sensory-autonomic neuropathy (HSAN), familial amyloid polyneuropathy (FAP), Tangier disease, and Fabry disease also exist.
Clinically, postural lightheadedness, dry mouth, dry eyes, impotence, loss of sweating or hyperthermia, nocturnal diarrhea, gastroparesis, impaired accommodation, urinary or bowel incontinence, and small fiber neuropathy are some of the presenting symptoms. Most peripheral neuropathies affect all fiber sizes. Few peripheral neuropathies are associated with pure or predominantly small fiber involvement. A large proportion is associated with diabetes. Painful burning feet is caused by a sensory neuropathy with small fiber involvement in more than 90% of cases. Patients with pure small fiber involvement display normal large fiber function. Muscle bulk, strength, muscle stretch reflexes, and large fiber sensory function (ie, vibration, proprioception) are normal.
Myelinated vs unmyelinated small fibers
Small fibers are both myelinated and unmyelinated. Small myelinated fibers transmit preganglionic autonomic efferents (B fibers) and somatic afferents (A delta fibers). Unmyelinated (C) fibers transmit postganglionic autonomic efferents as well as somatic and autonomic afferents. Both A delta and C fibers are widely distributed in skin and deep tissues.
The neurotransmitter for preganglionic sympathetic and parasympathetic nervous system (PNS) as well as postganglionic parasympathetic nervous system is acetylcholine (ACh). The neurotransmitter for the postganglionic sympathetic nervous system (innervating sweat glands) is also acetylcholine, whereas that for the remaining postganglionic sympathetic nervous system is norepinephrine (NE).
Electromyography (EMG) plays a key role in the evaluation of most peripheral neuropathies and helps in assessing only large myelinated fibers. Thus, pure small fiber neuropathies may be associated with normal findings on routine electrophysiologic studies. Elderly patients who lack sural sensory responses can still be diagnosed with small fiber neuropathy. Patients with symptoms other than neuropathic ones certainly need autonomic function testing for appropriate diagnosis.
The central autonomic network is a complex network in the central nervous system (CNS) that integrates and regulates autonomic function. The network involves the cerebral cortex (the insular and medial prefrontal regions), amygdala, stria terminalis, hypothalamus, and brainstem centers (periaqueductal gray, parabrachial pons, nucleus of the tractus solitarius, and intermediate reticular zone of the medulla). 
The afferent pathways have receptors residing in the viscera and are sensitive to mechanical, chemical, or thermal stimuli. They conduct along somatic and autonomic nerves and enter the spinal cord through the dorsal roots or the brainstem through cranial nerves. Impulses initiate local, segmental, or rostral reflexes.
The autonomic nervous system (ANS) consists of the sympathetic and parasympathetic nervous system. The sympathetic nervous system (SNS) descends to the intermediolateral and intermediomedial cells in the thoracolumbar regions of the spine, extending from TI to L2. Preganglionic axons exiting the spinal cord enter the white rami communicantes to join a network of prevertebral and paravertebral ganglia. These preganglionic axons are relatively short, myelinated, and cholinergic. Postganglionic axons exit the ganglia through the gray rami communicantes and extend with the peripheral nerves and blood vessels to innervate their end organs. These postganglionic axons are long, unmyelinated, and primarily adrenergic, except for the innervation of the sweat glands, which are cholinergic.
Adrenergic receptors are (1) alpha, which cause peripheral vasoconstriction; (2) beta 1, which increase heart rate and contractility; or (3) beta 2, which cause relaxation of smooth muscle located in the peripheral vasculature, bronchi, GI tract, and GU organs. The parasympathetic nervous system (PNS) exits the central nervous system primarily with cranial nerves III, VII, IX, and X, as well as the sacral spinal roots. Preganglionic axons are generally myelinated and have long peripheral projections before synapsing with postganglionic neurons in ganglia that are located close to the end organs; preganglionic axons are also cholinergic. The postganglionic axons are short and cholinergic; cholinergic receptors are also known as muscarinic receptors because of the pharmacology that defines them. 
Nerve fibers contributing to the superior hypogastric plexus and the hypogastric nerves are currently considered to comprise an adrenergic part of the autonomic nervous system located between vertebrae T1 and L2, with cholinergic aspects originating from sacral spinal segments S2-4. The illustrates the nature of the superior hypogastric plexus, which gives a better understanding of the urinary and sexual dysfunctions after surgical injuries. 
Cardiac and Vascular Regulation
The concept of central integration in cardiac and vascular regulation can be better understood by knowing that any increase in blood pressure and cardiac output increases the activity of the afferent pathway, which reflexly inhibits sympathetic activity or activates parasympathetic activity or both. However, any decrease in blood pressure and cardiac output decreases afferent activity, which reflexively increases excitatory responses. Thus, cardiovascular function is controlled by a negative-feedback system, and increasing activity of the afferent pathway results in decreasing activity of the sympathetic efferent pathway and/or increasing activity of the parasympathetic efferent pathway and vice versa.
In the afferent pathways, arterial baroreceptors located in the carotid sinus, aortic arch, and various thoracic arteries respond to changes in blood pressure and give rise to afferent activity, which conducts in the glossopharyngeal and vagus nerves. The cardiac mechanoreceptors are sensitive to mechanical deformation of the cardiac chambers and gives rise to afferent activity, which conducts in the vagus nerve. The pulmonary stretch receptors are sensitive to lung volumes, and inhalation increases afferent activity, which conducts in the vagus nerve.
In the efferent pathways, the sympathetic nervous system (SNS) is predominantly involved in cardiac and vascular regulation, and the parasympathetic nervous system (PNS) only has a little influence on the peripheral vasculature. Postganglionic sympathetic fibers innervate the atria, the ventricles, and coronary arteries from the cervical ganglia as the superior, middle, and inferior cardiac nerves or from thoracic ganglia at the TI-T4 level. Stimulation causes increased heart rate, increased myocardial contractility, and coronary vasodilatation.
Postganglionic sympathetic fibers innervate the vasculature from plexi on the large proximal vessels or from the somatic nerve. The innervation is denser in resistance vessels (small arteries and large arterioles) than in capacitance vessels (venules and veins). A balance of alpha adrenergic (vasoconstricting) and beta adrenergic (vasodilating) innervation exists. Preganglionic parasympathetic fibers innervate the atria, the ventricles, and coronary arteries from the vagus either by the superior and middle cardiac rami or by the recurrent laryngeal nerve as the inferior cardiac rami. Stimulation causes decreased heart rate, decreased contractility, and coronary vasoconstriction.
The window of opportunity for aggressive control of all the traditional risk factors for cardiovascular events or sudden death with intensification of therapy is with short-duration diabetes, the absence of cardiovascular disease, and a history of severe hypoglycemic events.  Autonomic dysfunction and neuropathy have become the most powerful predictors of risk for mortality.
Central integration of thermoregulation is controlled primarily in the preoptic and anterior hypothalamus, where a set-point is established by a balance between the activities of the thermosensitive neurons. When body temperature is below the set-point, autonomic reflexes generate heat by shivering and reduce convective heat loss by cutaneous vasoconstriction and piloerection. When body temperature exceeds the set-point, sudomotor activity stimulates sweating to increase evaporative heat loss and precludes cutaneous vasoconstriction and piloerection.
Afferent activity originates from thermosensitive neurons located within the hypothalamus, skin, abdominal viscera, spinal cord, and brainstem. Sleep-wake cycles, hormonal cycles, fluid balance, exercise, metabolic status, and humoral factors influence thermoregulation.
In the efferent pathway, thermoregulation is predominantly controlled by the sympathetic nervous system (SNS) with only a little involvement of the parasympathetic system (PNS). Sympathetic sudomotor fibers, which are the only sympathetic postganglionic fibers that are cholinergic, innervate the sweat glands to regulate evaporative heat loss.
Sympathetic vasomotor fibers cause vasoconstriction of cutaneous vasculature composed of abundant arteriovenous anastomoses in the dermis, which shunts blood flow away from the surface to reduce convective heat loss. The control of pilomotor function is rudimentary in humans, but contraction reduces surface area, which reduces convective heat loss.
Central integration for pupillary regulation is in the dorsal midbrain and Edinger-Westphal nucleus.
The afferent pathway is along the optic nerve.
In the efferent pathways, preganglionic sympathetic innervation of the pupil is from the C8-T2 spinal segments via the superior cervical ganglion. Postganglionic fibers extend along the carotid artery to the cavernous sinus and then enter the orbit with the fifth cranial nerve. The major action is pupillary dilation and also involves Mueller's muscle of the upper lid.
The parasympathetic innervation is from the third cranial nerve and ciliary ganglion and innervates pupillary constrictor muscles and the ciliary muscle for accommodation.
Central integration occurs in spinal centers and the central autonomic network.
The genitourinary system's afferent activity is along autonomic and somatic pathways.
Sympathetic innervation of the genitourinary system is from the T11-L2 spinal segments, the inferior mesenteric and superior hypogastric ganglia, and the hypogastric nerves. It causes uterine contraction, ejaculation in males, bladder wall inhibition, detrusor and trigone muscle contraction, and urethral smooth muscle contraction.
Parasympathetic innervation of the genitourinary system is from the S2-S4 spinal region and the pelvic nerves. It causes genital vasodilation, erections in males, bladder wall contraction, detrusor and trigone muscle relaxation, and internal sphincter relaxation.
The external sphincters are innervated by the pudendal nerve and are under somatic, not autonomic, control.
Central integration of the gastrointestinal (GI) system occurs in spinal centers and the central autonomic network, mainly the nucleus of the tractus solitarius and the nucleus ambiguus.
The afferent pathways synapse locally or in the ganglia, spinal cord, and more rostral portions of the autonomic nervous system.
The efferent pathways occur through the local integrative system, called the enteric nervous system, which consists of networks of nerves and plexuses embedded in the wall of the GI tract and integrated into local circuits for a variety of operations such as secretion, absorption, peristalsis, and sphincter coordination.
The sympathetic innervation is from the thoracolumbar segments by way of the celiac, superior mesenteric and inferior mesenteric ganglia, and the splanchnic, hypogastric, and colonic nerves. The main effects are stimulation of the esophageal sphincters and relaxation of the motility and internal rectal sphincter.
The parasympathetic innervation is from the vagus nerve, which innervates the esophagus, stomach, small intestines, and proximal colon and by the sacral outflow from S2 to S4, which innervates the distal colon and internal anal sphincter. The main effects are to stimulate motility and relax the internal rectal sphincter.
The external sphincters are innervated by the pudendal nerve and are under somatic, not autonomic, control.
Overview of Autonomic Testing
Various tests measuring autonomic function are available. [5, 6, 7] Both cardiovascular and sweat tests can be used to evaluate autonomic function. The sensitivity of the tests is variable according to the underlying disorder.
Most laboratories perform a battery of multiple tests to enhance reliability and sensitivity of various autonomic functions. A typical screening battery includes heart rate response with deep breathing (HRDB), Valsalva ratio and Valsalva maneuver analysis, orthostatic testing, and at least one of the available tests of thermoregulatory function.
Before testing, patients should abstain from alcohol, caffeine, and nicotine for 3 hours (preferably 12 h). Medications with anticholinergic properties (eg, antidepressants, antihistamines, and certain over-the-counter [OTC] medications), adrenergic antagonist action (eg, beta blockers), sympathomimetic properties, parasympathomimetic properties, and fluid-altering properties (eg, diuretics or fludrocortisone) should be stopped. Note that consultation with the patient's primary physician may be required to stop some of these medications.
Patients should be rested and relaxed during and before the testing. Compressive dressings such as elastic stockings should be removed.
Advantages/disadvantages of autonomic testing
Autonomic testing has several advantages, including verification of the diagnosis; precise neuroanatomic localization of the abnormality to the central or peripheral nervous system levels; prognosis for the severity, staging, and monitoring of the treatment; and determination of the physiologic organ systems involved and the predominant system involved, whether it's sympathetic, parasympathetic, or both.
Small, unmyelinated nerve fibers that control many autonomic functions are inaccessible for direct neurophysiologic recording. In addition, numerous technical and physiologic variables must be controlled.
Tests of Sweating
Quantitative sudomotor axon reflex test
The quantitative sudomotor axon reflex test (QSART) is the most sensitive test of distal small fiber neuropathy (see the images below). This test involves iontophoresis of acetylcholine (ACh) onto the skin to stimulate sympathetic C-fibers in the sweat glands. The sweat response that is evoked is quantitated using a sudomotor, which measures the humidity of the evoked sweating response. Generalized dysautonomias, complex regional pain syndrome, atopic dermatitis, anticholinergic medication use, and abnormalities of the skin and sweat glands can interfere with the test results. 
Thermoregulatory sweat test
In the thermoregulatory sweat test (TST), patients are placed in a warming cabinet to provoke sweating. Their sweating pattern is then assessed by the color change of alizarin powder dusted over the body, limbs, and forehead (see the following image). 
Sympathetic skin response test
The sympathetic skin response (SSR) test is based on the fact that electrodermal activity reflects sympathetic cholinergic sudomotor function, which induces changes in resistance of skin to electric conduction.  Many modalities of stimulation suffice to elicit the potential reflexly, including electrical depolarization of a sensory nerve in the digit that startles. Other eliciting stimuli include a startling auditory sound or deep inspiratory gasps.
The potentials in the hands have larger amplitudes and shorter latencies than those in the feet. The latency is about 1.5 seconds in the hand and about 2 seconds in the foot following an eliciting stimulation. The major contributor to latency is the efferent conduction along the sudomotor pathways, which are small, unmyelinated C fibers.
Tests of Cardiovascular Function
Head-up tilt table testing
Beat-to-beat blood pressure measurements formerly required invasive intra-arterial recording, but modern photoplethysmographic (Finapres) devices generate waveforms similar to intra-arterial recordings and allow noninvasive recording.
Postural physiology has been studied in the laboratory by head-up tilt on a tilt table.  Upon changing from a recumbent to upright position on a tilt table, there is almost one third of a shift of venous blood from the central to the peripheral compartment; approximately 50% of the change occurs within seconds. This results in decreased cardiac filling pressures and the stroke volume is decreased by up to 40%, which decreases afferent activity from the sensory baroreceptors. The heart rate rises, first from withdrawing parasympathetic activity and then from increasing of sympathetic activity. Overall, the cardiac output only drops 20%, and blood pressure is largely maintained. See the image below.
Cardiovascular responses to standing and 30:15 ratio
Standing induces an exercise reflex as well as mechanical squeeze on both venous capacitance and arterial resistance vessels. The changes stimulate the baroreceptors; a pronounced neurally mediated reflex ensues, which decreases sympathetic outflow, releases vasoconstrictor tone, decreases total peripheral resistance by up to 40%, and drops blood pressure by up to 20 mm Hg; these changes last 6-8 seconds.
The heart rate increases immediately upon standing and continues to rise for the next several seconds.  The initial cardiac acceleration upon standing is an exercise reflex that withdraws parasympathetic tone, and subsequent changes are baroreflex-mediated changes, which enhance sympathetic tone.
Heart rate variation with deep breathing
The variation of heart rate with respiration is known as sinus arrhythmia and is generated by autonomic reflexes (see the following image).  Inspiration increases heart rate, and expiration decreases it. The variation is primarily mediated by the vagus innervation of the heart. Pulmonary stretch receptors as well as cardiac mechanoreceptors and possibly baroreceptors contribute to regulating the heart rate variation. It increases with slower respiratory rates and reaches a maximum around 5 or 6 respirations per minute.
Valsalva maneuver and Valsalva ratio
The Valsalva maneuver consists of respiratory strain that increases intrathoracic and intra-abdominal pressures and alters hemodynamic and cardiac functions (see the image below). The Valsalva maneuver is usually recorded by invasive monitoring of the intra-arterial blood pressure. Levin monitored the heart rate alone without monitoring blood pressure during the Valsalva maneuver and calculated a ratio of the fastest heart rate to the slowest as a way of noninvasively quantifying the procedure.  Newer photoplethysmographic monitoring devices are able to noninvasively record beat-to-beat blood pressures as well as heart rates, thus allowing easier evaluation of the maneuver. [1, 15]
The Valsalva maneuver has the following 4 phases:
Phase I: This phase is transient and lasts only a few seconds, with increase of the blood pressure caused by increased intrathoracic pressure and mechanical squeeze of the great vessels
Phase II: This phase has early and late components; in early phase II, venous return decreases, which results in decreasing stroke volume, cardiac output, and blood pressure; in about 4 seconds, in late phase II, blood pressure recovers back toward baseline levels; this recovery stems from increased peripheral vascular resistance from sympathetically mediated vasoconstriction
Phase III: This phase occurs with release of the strain, which results in a transient, few-seconds-long blood pressure decrease caused by mechanical displacement of blood to the pulmonary vascular bed, which had been previously under increased intrathoracic pressure
Phase IV: This phase occurs with further cessation of the strain; the blood pressure slowly increases and heart rate decreases; because the blood pressure rises to above baseline levels and the heart rate to below baseline levels, it is often called the "overshoot"
The Valsalva ratio is the ratio of the maximal heart rate in phase II to the minimal heart rate in phase IV. This may be calculated easily as the ratio of the longest R-R interval during phase IV to the shortest of phase II.
Blood pressure response tests
Blood pressure response to sustained handgrip
Persistent muscle contraction causes blood pressure and heart rate to increase. The mechanism involves the exercise reflex, which withdraws parasympathetic activity and increases sympathetic activity. This test requires the patient to apply and maintain grip at 30% maximal activity for up to 5 minutes; the diastolic blood pressure should rise more than 15 mm Hg. 
Blood pressure response to mental stress
Mental stresses such as arithmetic, emotional pressure, and even sudden noise can cause sympathetic outflow to increase, which leads to increase in blood pressure and heart rate. This test has been used as a measure of sympathetic efferent function that has the advantage of not requiring direct afferent stimulation. 
Blood pressure response to cold water immersion
In 1932, Hines and Brown noted an increase in blood pressure after submerging a patient's hand in ice water. The afferent limb of the reflex is somatic, and the efferent limb is sympathetic.
Many patients find that maintaining the hand in ice water for the requisite time period is difficult. This test also lacks sensitivity, as many normal subjects do not have a significant rise of blood pressure.
Plasma catecholamine levels and infusion tests
Plasma norepinephrine levels approximately double with the upright posture in view of initiation of vasopressor responses, which are sympathetic and adrenergic. In preganglionic sympathetic disorders such as multisystem atrophy, resting supine norepinephrine levels are normal but fail to rise when at standing because of the lack the preganglionic drive. In postganglionic sympathetic disorders, such as progressive autonomic failure, resting supine norepinephrine levels are low and fail to rise when at standing. [18, 19]
Tests of pupillary regulation
When parasympathetic denervation exists, denervation hypersensitivity occurs, and the pupil constricts to such dilute stimulation. Similarly, epinephrine acts directly on sympathetic adrenergic dilatory muscles to cause pupillary dilatation. In very dilute amounts (0.1% solution), however, it normally causes minimal dilatation. When sympathetic denervation exists, denervation hypersensitivity occurs, and the pupil dilates. Cocaine (4-5% solution) blocks reuptake of norepinephrine in sympathetic nerve terminals innervating pupillary dilator muscles and causes pupillary dilatation. 
Tests of gastrointestinal autonomic regulation
Manometry or pressure transducers placed in different portions of the gastrointestinal (GI) tract help localize sites of stasis. Sympathetic denervation may be identified by various neurochemical studies, including the norepinephrine response to edrophonium.  Parasympathetic denervation may be identified by the plasma pancreatic polypeptide response to sham feeding or hypoglycemia. In multiple system atrophy, the rectal sphincter is frequently denervated from degeneration of the Onuf nucleus in the sacral spinal cord,  and electromyography of the rectal sphincter may be abnormal in these patients.
Tests of genitourinary autonomic regulation
Electrophysiologic tests such as bulbocavernosus reflexes, sensory conduction in the dorsal nerve of the penis, pudendal sensory-evoked potentials, motor latencies of the pudendal nerve, and routine and single fiber electromyography of the sphincters test somatic, not autonomic function. Methods of electrophysiologic study of the smooth muscle of the corpus cavernosum have been used as well. 
Yuan et al recently identified a novel homozygous mutation in SCN9A from 2 Japanese families with autosomal recessive hereditary sensory autonomic neuropathy.  This loss-of-function SCN9A mutation results in disturbances in the sensory, olfactory, and autonomic nervous systems.