NEUROTRANSMITTER SYSTEMS

 

Catecholamines; synthesis and storage

 
 

The catecholamines that act as neurotransmitters include dopamine and norepinephrine. Epinephrine is not produced in the central nervous system, but in the peripheral nervous system (adrenals). The catecholamines share a common synthetic pathway. The rate limiting step is tyrosine hydroxylase. The activity of tyrosine hydroxylase can be modified by phosphorylation. This provides a point of regulation for the neuron. Clinically there are no agents capable of modulating tyrosine hydroxylase. However, treatment with agents such as methyldopa can compete with dopa for further processing. The result is a formation of false neurotransmitters. The false neurotransmitters are packaged in the synapse as though they were the catecholamine but when released into the synapse they are ineffective at the receptor. False neurotransmitters such as octopamine are also thought to be increased in hepatic encephalopathy. The packaging of false transmitters in the periphery is thought to be increased by inhibiting MAO. This is thought to explain the orthostatic blood pressure effects caused by therapeutic doses of MAO inhibitors.

 

 

The major biochemical difference between the norepinephrine and dopamine neurons is the presence of a vesicle bound copper containing enzyme in the neurons of the norepinephrine neurons. This enzyme is dopamine beta hydroxylase. It catalyses the synthesis of norepinephrine from dopamine.

 

The catecholamines have similar metabolic pathways. The chart below shows these pathways. A point of potential therapeutic intervention in metabolism is at the level of monoamine oxidase. MAO is primarily found associated with the mitochondria. The intracellular site of MAO suggests it is involved in metabolizing catecholamines once they have been taken back up into the neuron to prevent repackaging them into the synaptic vesicles. There are two MAO isoenzymes. MAO-A appears to be a specific enzyme for serotonin and norepinephrine. MAO-B is a more broadly active enzyme that acts on phenylethylamines.

Tyramine and dopamine are equally good substrates for both MAO-A and MAO-B. There is an anatomic distribution of the isoenzymes. MAO B is found primarily in the brain. MAO A is found thought the body and especially in the GI tract where it may serve to limit the absorption and physiologic effect of dietary monoamines. There are specific inhibitors of each of these. Nonspecific MAO inhibitors must inhibit all the MAO. Because specific MAO inhibitors only inhibit one of the isoenzymes there is always MAO activity present. This has a couple of consequences; first there is less chance for a tyramine effect, second there is less orthostatic effect.

 

 

As you see from the chart the catecholamines are also metabolized by catechol-O-methyl transferase (COMT). COMT is present extracellularly. MAO can act before or after COMT of the catecholamines. Make note of the metabolic products as these can reflect the turnover of the neurotransmitters.

 

Catecholamine synapse

 

The norepinephrine and dopamine synapses are basically analogous. Important extracellular features include the presence of reuptake sites, multiple post synaptic receptors, and autoreceptors and presynaptic receptor sites. The 'function' of a neurotransmitter depends on a combination of the network it is being used in, and the post-synaptic receptor's behaviors. The autoreceptors modulate the release of neurotransmitter by the presynaptic neuron. The autoreceptors are believed to have a difference in affinity with the neurotransmitter. This is shown by the fact that extremely low doses of a receptor agonist are inhibitor to the release of endogenous transmitter and low dose of a receptor antagonist augments release of neurotransmitter. It is believed that this explains the early increase in HVA that occurs when neuroleptics are first initiated. Some neurons appear to have both presynaptic facilatory and inhibitory receptors. The presence of both a presynaptic facilitating and presynaptic inhibitory autoreceptor suggest the need for fine tuning of the release of the neurotransmitter. It should be obvious that the relative proportion of each can adjust the set point of the neurons release of neurotransmitter much like the max and mins of a thermostat adjust the activity of a furnace in the home. Enduring changes in activity can be acheived by altering the genetic expression of one or both of the genes for these autoreceptors. In general the mechanism of the effects of these presynaptic autoreceptors are the same as in presynaptic inhibition or facilitation. That is they modulate Ca++ channels or influence the membrane potential of the presynaptic neuron or they influence adenylate cyclase activity.

 

Catecholamine projections

 

The behavioral effect of a pharmacologic intervention on a specific neurotransmitter is related to a number of things. An idea of the behavioral effects of the catecholamine pathways are disclosed by considering the sites of projection. The diagrams below illustrate the projections of the dopamine systems and norepinephrine systems in the human brain.

 

There are two major cell groups of norepinephrine containing cells in the neuroaxis. The First arises from the locus ceruleus in the caudal pontine grey and spreads anteriorly forming an extensive net throughout the cortex. The second cell group projects from the lateral tegmental neurons proceed caudally to the spical cord and rostrally into the diencephalon. The axons of the LC neurons have varicosities which allows a widely distributed release of norepinephrine. It is felt that this reflects a neuromodulatory function of this system. There are two norepinephrine tracts relevant to behavior which lead out of the LC and lateral tegmental neurons; the dorsal bundle and the median forebrain bundle. The widespread network of these neurons includes innervation of specific hypothalamic and thalamic cell groups. There are several theories of the role of the locus ceruleus in learning, memory, anxiety and psychosis. A more general and more understandable role can be described as a system of the orientation of the brain to stimuli in the environment and viscera. This system is activated by a variety of sensory stimuli. It seems to be related to vigilance. Such orientation response is needed to explore the environment. It is a necessary part of the central control of the autonomic nervous system. This system is also a part of the sleep-wake system as we will discuss in a subsequent section. Overactivity of the locus ceruleus system is implicated in anxiety disorders and drug withdrawal states. Another aspect of the vigalence system is its role in reward and reinforcement. It can be seen that dysfunction of the LC would result in hypothalamic dysfunction, anxiety, hedonic alterations, autonomic arousal, and sleep disturbances. It is little wonder that the LC is a focus of interest in affective disorders, anxiety disorders, and drug addiction and withdrawal.

 

The fibers of the NE tract sweep over the anterior pole before proceeding caudally. Thus, lesions along the pathway can result in a functional decrease in NE activity. This is believed to be part of the reason that anterior strokes result in a state similar to functional depression.

 

The fibers from the Lateral tegmental neurons proceed caudally into the spinal cord and anteriorly into the deincephalon and basal forebrain region. The basal forebrain is the region just inferior to the anterior part of the corpus callosum. This is an extremely important region behaviorally. It includes the septal nuclie which are important in the reward and reinforcement system .

 

 

The projections of the dopamine system comprise five basic systems. Four of these are behaviorally relevant. These include the mesocortical, mesolimbic, tuberoinfundibular and nigro striatal systems. The mesocortical, mesolimbic, and nigrostraital systems have several similarities pharmacologically. In general the nigrostriatal system is concerned with the initiation and maintanence of motor behaviors. It runs from the substatia nigra to the caudate and putamen. There is a feedback inhibition loop which includes acetylcholine, and GABA. The relevance of this loop is apparent in situations where it is interupted.

 

The mesolimbic and mesocortical systems arise from the ventral tegmentum. This cell group is barely separable from the substantia nigra. The mesolimbic system projects to elements of the limbic system including the amygdala, hippocampus, nucleus accumbens and the spetal area. Of significance these areas are in close proximity to the caudate and putamen. In fact some neuroanatomist call this region the limbic striatum. The mesocortical system projects to the frontal cortex. There appears to be separate feedback loops from these systems. Because of the intermingled nature of the cells of origin a feedback in the mesolimbic system also affects the mesocortical system and vis versa. The result of this situation will be considered when we discuss schizophrenia.

 

The tuberoinfundibular tract has a major role in the regulation of some hypothalamic and pituitary peptides. Principle among these is prolactin. Inhibition of dopamine activity in this tract results in an increase in prolactin release. This explains the galactorrhea seen with neuroleptic use. Of more research interest is the potential use of prolactin to monitor central dopamine blockade. Some speculate that lack of a prolactin increase with neuroleptics predicts treatment non-response. Others have not seen such a correlation. Another aspect of this system is that it is inhibited in acute stress. This make prolactin one of the so-called stress hormones. This is the reverse of activity in the mesocortical and mesolimbic system which is activated in stress. Other important differences is the lack of autoreceptors on the presynaptic terminals inthe tuberoinfundibular system. The result is that although similar in many ways theinfundibular tract differs pharmacologically and for that reason may not be a particularly good predictor of response in the mesolimbic/mesocortical systems.

 

The mesolimbic and mesocortical systems appear important in the initiation and maintenance of goal directed and reward mediated behaviors. This includes the proper maintenance of cognitive sets (ie logical thought). A dysfunction in this system alters the normal association process and leads to a breakdown in the proper perceptual functioning of the heteromodal areas of the frontal lobe . This results in an inability to screen nonmeaningful stimuli. Possible consequences would include such experiences as loosening of associations, bradyphrenia, flight of ideas, delusional perceptions. In addition there may also be a role of the dopamine system in regulation of affective expression. A classic disease state of increased dopaminergic activity is Huntington's Disease. Parkinson's disease is the classic example of dopamine deficiency. Although these diseases are usually considered movement disorders they have parallel effects on the limbic striatum as well. In fact these diseases are often missed when the first presentation is to a psychiatrist.

 

Dopamine receptor dynamics

 

There is good evidence for at least four subtypes of postsynaptic dopamine receptors. The D1 and D2 receptors utilize differing transduction mechanisms. D1 appears to be linked to cAMP via a G-s unit. D2 appears to be linked via a G-i unit. D2 also inhibits Ca++ entry through the voltage sensitive channels. There is also evidence that D2 increases K+ conductance and leads to a hyperpolarized state. This would inhibit the post synaptic neuron. Both D1 and D2 appear to have an influence on the IP3 system. A subtype of the D2 receptor is the D4 receptor. This D4 receptor has a greater affinity for clozapine. It is distributed in the frontal cortex, midbrain and amygdala. The D4 receptor appears to be in low concentrations in the motor striatum. This may help explain the general lack of EPS with clozapine.

 

 

Serotonin; synthesis and release

Serotonin is an indolamine monoamine neurotransmitter. The synthetic pathway is analogous to the catecholamines in many ways. An important distinction is that the rate limiting step is the uptake of tryptophan into the neuron. Tryptophan availability is the actual rate limiting factor in the intact animal. Tryptophan crosses the blood brain barrier via an active transport mechanism in competetion with other neutral amino acids such as leucine, lysine, and methionine. The activity of this transport mechanism is facilitated by the presence of insulin and glucose. Another interesting aspect of this system is the fact that tryptophan is one of the few amino acids which is bound in the plasma to any significant degree. The actual binding site is the fatty acid binding site of the albumen. This system allows a multitude of factors to ultimately influence the rate limiting step in serotonin synthesis. For example anything which increases free fatty acids would displace the tryptophan and thus increase the percent free which is able to cross the BBB. An example of such events include any acute stressor which increases glucocorticoid response, exercise, and acute alcohol consumption.

 

The metabolism of serotonin is primarily done by MAO. The prinicple metabolite is 5HIAA. The same statements concerning the CSF measurement of MHPG and HVA applies to 5HIAA.

 

Serotonin receptors

 

There are three basic types of serotonin receptors; 5HT-1, 5HT-2, and 5HT-3. The 5HT-3 receptor is present in the area postrema which stimulates emesis. The 5HT-1 and 5HT-2 receptors are of a greater interest for psychiatry. The 5HT1 receptors have been subtyped by DNA cloning and differential pharmacology into four major subtypes. The most important is the 5HT-1a which is located in the Raphe and Hippocampus. This receptor is implicated as an autoreceptor which modulates 5HT release from presynaptic neurons. In addition the 5HT-1a receptors are G-protein linked and have been implicated in thermoregulation, arteriolar vasomotor responses, hypotension, sexual behavior, and possibly sleep. The 5HT-2 receptors are located throughout the cortex and have been implicated in platelet aggregation, vasomotor contraction, head twitches, and possibly sleep. Pharmacologically these receptors are important and are affected by a wide variety of pharmacologic agents including butyrophenones, and phenothiazines.

 

The projections of the serotonin system

Note that they arise from the dorsal raphe and the raphe magnus. These nuclie are situated to observe ascending sensory input and act to facilitate information processing. One example of this is in the slow wave sleep. We will discuss this later when we discuss sleep and sleep disorders. Other implications of the sensory input into these nuclie are the concepts of sensory gating and directed attention. Recall that the Locus Ceruleus is important is arousal and vigilance. Vigilance is a state of increased arousal. Vigilance is necessary for focus or directed attention but not suffecient. Focused attention requires that incoming sensory information be given a priorty according to importance. The process of habituation occurs if stimuli is not reinforced. A lack of directed attention can appear as impaired concentration.

 

Disrution of the normal serotonergic tone in animals can affect their exploratory behavior. Animals seem to endow meaningless stimuli with relative behavioral importance. That is they seem to have perseverative responses in serotonin defiecient states and behavioral over activity in states of serotonin excess. It has been suggested that some patients with neuroleptic resistant psychosis have a dysregulated serotonin system. Some of the effects of atypical neuroleptics are thought to be mediated through a 5HT antagonism. It would seem that these agents restore a proper sensory gating and ability to direct attention

 

Acetylcholine, synthesis and release

 

Acetylcholine is one of the oldest and best understood neurotransmitters. This is likely because of its actions at the neuromuscular junction and the accessibility of this site to study. Unfortunately the ability to study the central cholinergic system is relatviely new. Unlike the catecholamines acetylcholine is a relatively simple structure with no aromatic rings. The ability to measure acetylcholine in body fluids had to await the development of better assays in the late 60's and 70's.

 

 

The synthesis of acetylcholine is relatively simple and straightforward. It is sythesized from acetylCoA + choline by an enzyme known as choline acetyltransferase (CAT). This enzyme is contained only in cells which synthesize acetylcholine. It is a marker enzyme which identifies cholinergic cells. Recall that Acetyl CoA is derived during the metabolism of glucose. Choline is derived from deitary sources and from phosphatidylcholines. After acetylcholine is released into the synapse it ultimately undergoes hydrolysis to release the choline and acetate. The choline is taken back up into the presynaptic neuron about 1/3 to 1/2 of the time. The choline can then be used ot resynthesize acetylcholine or can be used to synthesize phospholipids which can be used as stores of choline. The diagram below describes the points of pharmacologic interventions.

 

Acetylcholine is metabolized by cholinesterases. There are several types the two most often referred to in reference to acetylcholine metabolism are acetylcholinesterase and butyrylcholinesterase or pseudocholinesterase. In the synapse the cholinesterase seems to be intimately related to the actual receptor. This enzyme is inhibited by physostigmine and organophasphates.

 

Acetylcholine receptors

 

There are two major types of cholinergic receptors based on differential binding. The first type is the nicotinic receptor and the second is the muscarinic receptor. DNA cloning has identified five subtypes of muscarinic receptors. All of these muscarinic receptors are G-protein linked. The muscarinic subtypes also seem to have a differential distribution in mammalian brain. The nicotinic receptor is a ligand gated channel composed of five subunits. It is primarily the responsible for the peripheral effects of acetylcholine at the autonomic ganglia and the neuromuscular junction. There may be some nicotinic receptors centrally but their contribution is uncertain.

 

The nicotinic receptor is composed of 5 subunits. Of interest is the finding that injection of one of the more lipophilic subunits into the rabbit results in a syndrome indistinguishable from myasthenia gravis. This has confirmed suspicions that MG is an autoimmune disease.

 

Nuclear medicine techniques are able to preferentially bind the muscarinic receptors with an agent known as dexitimide and QNB. Studies are underway for evaluating the sensitivity of these agents in the evaluation of Alzheimer's disease.

 

Acetylcholine projections

The chart below illustrates the major central projections of the cholinergic system. In essence there are hree important cholinergic systems for neuropsychiatry. The first is the nucleus basalis of Meynert. This nucleus lends projects to the cortex. It is one of the nuclie which is impaired in senile dementia of the Alzheimer's type. It is suggested that this nuclie plays a role in learning and memory. In addition the cholinergic neurons in the basal forebrain are involved in cognitive intergration of vegative and motivationally relevant information. We will discuss this in more detail when we discuss the dementias.

 

The second system arises from the brainstem. This system sends cholinergic fibers to the midbrain and thalamus. It is suggested that this system is related to sleep-wake rhythms and turning on REM. It is felt that this group of neurons acts as a sensory filter in some way. It has been shown, (here at UAMS) that schizophrenics has a less CAT in the pontine cholinergic nuclie. The meaning of this is uncertain. It is of interest that anticholinergic agents are well known psychotomimetics in high doses.

 

The third group is the cholinergic neurons in the basal ganglia. These will be discussed in more detail in later lectures on movement disorders.

 

Gamma amino butyric acid, synthesis, storage and release

 

GABA was identified in the mammalian brain in 1950's. It is believed to be the major inhibitory neurotransmitter in the brain. It is this role which is of interest to the neuropsychiatrist. GABA is synthesized from Glutamate as shown below. The marker enzyme is Glutamic acid decarboxylase (GAD). GAD is a pyridoxal cofactor dependent enzyme. A congential form of B-6 vitamin deficiency is known to predispose to seizures which are B-6 responsive. Glutamate is a pivotal amino acid in the brain. It is dervied from alpha keto glutarate which is one of the intermediates in the Krebs cycle by way of the addition of an amine group. Glutamate also undergoes transamination to form glutamine by addition of another amine group. Glutamine then proceeds to the liver where it is deaminated to regenerated glutamate which then returns to the brain. This is brain's nitorgen cycle. In situations where the liver is unable to deaminate the glutamine the brain must obtain glutamate by draining the Kreb's cycle intermediates. This in turn begins to impair cerebral energy metabolism.

 

Following release GABA can be taken back up by the neurons or by astrocytes. It appears that the release of GABA is also under autoreceptor control. GABA is metabolized by the enzym GABA transaminase (GABA-T) to form succinic acid semialdehyde. Succinic acid semialdehyde is metabolized further to form succinic acid which is also a Kreb's cycle intermediate. GABA-T is inhibited by valproic acid. This is the basis for the belief that valproic acid is GABAergic. There are other alternative pathways for GABA metabolism.

 

GABA receptors

 

There are two basic subtypes, GABA-a and GABA-b. GABA-a is the most prevalent in the mammalian brain. The GABA-a receptor is similar to acetylcholine receptor in that it is related to an ion channel. In the

case of GABA-a it is the chloride ionophore. Binding of GABA to this receptor increases the permeability to chloride ion which causes a hyperpolarization of the neuron or inhibition. The GABA-a receptor has four basic subunits, 2-alpha and 2 beta peptides which surround a chloride channel. There are three basic binding sites on this complex. The first is the GABA site. The second is a benzodiazepine site. The third is in the channel and is essentially a barbiturate site.

 

Binding to the benzodiazepine site can have three effects, agonism, inverse agonism, or antagonism. The typical anxiolytic and sedative hypnotic agents such as diazepam and lorazepam act as agonist at these receptors. Their binding increases the affinity of the GABA binding site for GABA. This results in an increase in Cl- influx. Inverse agonism occurs with the beta carbolines. These agents act in the opposite direction as the agonist. Binding of these agents reduces the influx of Cl- below the baseline state. Clinically this is accompanied by anxiety. The antagonist such as flumazenil act to displace the agonist and

inverse agonist without a direct effect on the chloride channel it's self.

 

There is much speculation and a little evidence for an endogenous benzodiazepine ligand. It has been found to be increased in some metabolic conditions such as hepatic encephalopathy. In fact there are case reports of the use of benzodiazepine antagonist in the treatment of hepatic encephalopathy. Some patients have been noted to become less somnolent and more oriented.

 

It remains to be seen whether flumazenil will have any use beyond reversing benzodiazepine overdoses.Clinically agents which are GABA-A agonist are generally anticonvulsant in activity as well as muscle relaxants.

 

The GABA-b receptor is a G-protein related receptor which is distinct from the GABA-a sites. The highest concentrations of GABA-b receptors is in the interpeduncular nuclie and cerebellum. It appears that one of its prinicple effects is to increase the efflux of K+ from the cell. This would result in a hyperpolarization. Pharmacologically baclofen is considered a GABA-b agonist. The principle effect of GABA-b agonism is muscle relaxation.

 

A significant relationship of dopamine and GABA exists. In general GABA acts to reduce the firing of the dopaminergic neurons in the tegmentum and substantia nigra. It forms the basis for the use of benzodiazepines as augmentation strategies in the treatment of psychosis. In addition benzodiazepines may be helpful in cases where there is an over activity of dopamine in the motor striatum such as Huntington's Chorea or Tardive Dyskinesia. It is believed that they act by increasing the feedback inhibition. The feedback inhibition from the GABA neurons of the globus pallidus and putamen to the dopaminergic neurons of the substantia nigra is an important modulating force on the activity of the dopamine neurons.

 

Excitatory amino acids

 

Glutamate is considered the principle excitatory amino acid in the CNS. Its role in cellular metabolism is well known. Glutamate is distributed widely throughout the neuroaxis. Regions in which it seem particularly important include the granular cells of the cerebellum, the pyramidal cells of the hippocampus, the Betz cells of the motor strip, and the projections of the frontal lobe to the basal ganglia. One of the major difficulties in recognizing the role of glutamate as a neurotransmitter was the fact that there is only a small percentage of the glutamate present in synaptic vesicles. The vast majority of glutamate is present as part of intermediary metabolism. The ability to define the role of glutamte came as a result of finding receptors for glutamte and labeling them with various analogs.

 

There are four glutamate receptor subtypes of importance. More will undoubtably be found and the nomenclature may change in the next few years. The four receptor subtypes include the NMDA receptor, the quisqualte receptor (also called the AMPA receptor), the kainate receptor, and the metabotropic receptor. A common feature of these receptors is depolarization of the membrane potential. In addition there are some special characteristic which make this system interesting. First the receptor systems seem to have a degree of cooperativity. By this I mean that they function most completely as a group. The diagram below illustrates these resceptor systems.

 

Note that the NMDA receptor is present in an inactive state with a Mg++ ion blocking the calcium channel. For the NMDA receptor to become active the Mg++ must leave the channel site. This is accomplished by membrane depolarization brought about by the other glutamate receptors. Once this is done and the NMDA receptors are active a process known as excitotoxicity occurs. This may provide the basis for kindling and other longterm changes which result in neuronal specialization. Excitotoxicity is due to the opening of the calcium channels which results in an increase in free calcium. The degree of damage seems to be limited in most situations by an energy dependent mechanism of binding the calcium. In situations of ischemia or hypoglycemia there is a loss of this system and an excessive excitotoxicity.

 

Neuroscience has attempted to finding specific NMDA receptor blockers to protect against ischemic damage. Some of the compounds found to date include the benzomorphans, MK801, and phencyclidine. Each of these compounds has psychotomimetic activity. This has limited there utility to date. Focus is no on blockers at other domains of the molecule such as the calcium channel itself. Another potential site suggested by the diagram is the glycine site. Glycine acts as an allosteric facillitator. Binding of glycine increases the ability of glutamate to activate the channel apparently. It is unclear if this is the site that Nitric oxide acts.