Table of Contents

Nerve Physiology

Nerve Impulse: History

The spread of the electric impulse in the nerve fibres was speculated but only confirmed by a famous scientific experiment performed by a man who gave his name to one of the types of electricity. Galvani who comes from Bolognia performed the experiment in 1791. He connected two electrodes from a “Galvanic Battery” to a muscle of a recently killed frog. The muscle contracted when the current passed through the electrodes and returned to its previous state when the current stopped. Many secondary school biology students perform the same experiment in their classes nowadays. In a science fiction like Frankenstein, even a corpse can be brought back to life by an electric current.

NERVE CONDUCTION: HISTORICAL

|1791 |L. Galvani| frog muscle will contract when stimulated electrically.| |1850 |H. von Helmholz| speed of conduction in frog nerves is 40m/sec, ie it is not simply electrical.| |1856 |R Virchow | described neuroglia i.e. "nerve glue"| |1877 |du Bois Reynard |On the basis that curare blocked conduction to muscles, he proposed that nerve conduction was chemically mediated.| |1904| Elliott |showed that the effects of some neurones could be mimicked by adrenergic (from the adrenal) substances (sympathetic nervous system)| |1906 |H. Dale |showed that the effects of some neurones could be mimicked by muscarine compound(Ls (parasympathetic nervous system). This was followed the description of nicotinic effects. | |1924| F G Donnan |formulated the so called "Donnan Equilibrium" - this provided an explanation of chemical and charge effects across membranes when a large impermeable organic ion is present.| |1927 |W. Nernst |"Nernst Potential" Allowed calculation of membrane potentials from ion concentrations inside and outside a cell| |1930|Finkleman |Using a 2 heart superfusion preparation, demonstrated chemical transmission. i.e. the superfusate from a stimulated frog heart was shown to alter the activity of an unstimulated heart.| |1935| Dale's Principle | a neurone releases the same transmitter at all its synapses.| |1952 |Hodgkin–Huxley model | Squid axon studies - established the relationship between Na+/K+ fluxes and membrane depolarisation.| |1976 |Neher, Sakmann and others |Single channel currents recorded from membrane of denervated frog muscle fibres i.e. "patch clamping"| |1981| CoIquhoun, Sakmarin and others| Measurement of fluctuations in the microsecond time range of the current through single acetylcholine receptor linked ion channels- studies of this type showed that channels may open and close many times during the brief period of receptor occupancy by a agonist.| |1982| Cull-Candy, Parker and others. | Measurement of the rapid kinetics of opening of channel associated with glutamate receptors le evidence was obtained which demonstrated that amino acids could have a role in brain which was unrelated to protein synthesis.|

CLINICAL NATURE OF SYNAPTIC TRANSMISSION:

HISTORICAL

During the 19th century, several investigators such as Galvani, Volta and Fontana identified electrical activity in neuronal tissues. In 1897 Sherrington, first used the word "synapse" Elliott in 1904 suggested that sympathetic neurones release adrenaline. In 1906 Dale proposed that parasympathetic neurones release a muscarine-like substance.

Evidence for conduction between nerves being chemical rather than electrical came from several studies. 1. The time delay in conduction between nerves suggested a non electrical event. 2. Neuronal responses could be inhibitory. 3. No contra flow of electrical impulses between neurones were observed. 4. Amplification of signals could occur.

As mentioned above, the definitive evidence came in 1925 from Loewi's study which demonstrated that a superfusate from a stimulated frog's heart could cause contractions in a second heart preparation.

MEMBRANE POTENTIALS

When two electrodes are placed such that one is inside and the other is outside a cell it can be seen that a potential difference exists such that the inside of the cell is close to -5OmV with respect to the outside: this degree of polarisation exists in virally all cell types.

Membrane potentials exist and are maintained for the following reasons: - Organic acids (HA) in the neuronal cytoplasm dissociate to form H + and A-. - A- cannot freely diffuse out of the cell. - Intracellular Na+ ions are low relative to the external concentration (12OmM) due to the cell membrane being relatively impermeable to this ion. - The cell has high permeability to K+ and Cl- - The resting membrane potential is largely dependent on K+, i.e. it is the balance between the electrostatic and the concentration gradients for this ion which is approximately -7OmV (EK). The inside of the cell is relatively negative. Hence, the resting membrane potential of a cell can be shifted by increasing extracellular K+ and this fact is used in many experimental protocols.

Notes

  • The transport of Na+ across membranes is much lower than K+ because it is highly hydrated and because of the Na + pump which moves Na + out of cells.
  • The membrane potential can be calculated from the Nernst Equation, if the ionic concentrations are known. In fact, a more appropriate equation is the Goldman Constant Field Equation which involves ionic permeabilities rather than concentrations.

ACTION POTENTIALS

Neurones differ from other cells in that they are excitable i.e. unlike a normal cell when a nerve cell membrane, such as that of the giant axon of the squid, is depolarised from its resting value (- 7OmV) to -10 to 15mV, a rapid self-limiting process occurs by which the transmembrane potential is reduce and overshoots zero, so that the inside of the membrane becomes positive relative to the outside.

This is an action potential. When the cell begins to depolarise from stimulation current, the current flow across the membrane is carried by K+. As the membrane becomes more depolarised, the resistance to Na+ decreases and more enter, the cell along its electrical and chemical gradients. As Na+ enters, the membrane becomes even more depolarised. which further increases the conductance to Na+. These changes are voltage-dependent. and self-perpetuating. They are driven by the flow of Na + towards its equilibrium distribution, which should be proportional to its original extracellular and intracellular concentrations. However, the Peak of the action potential does not attain the equilibrium potential predicted on the basis of transmembrane Na+ concentrations because of a second phase of events.

The voltage-dependent increase in sodium conductance and the consequent depolarisation also activates a voltage-dependent K + conductance, which causes potassium efflux along its concentration gradient. This maintains the inside negativity of the cell and begins to reduce the membrane conductance to sodium, thus making the action pontential a self-limiting phenomenon. There is also calcium current i.e. due to Ca+ influx into the cell. However, although calcium entry into the cell can precipitate a multitude of events, It does not contribute significantly to the action potential. In most axons, the action potential last for 0.2 - 0.5ms. Reestablishment of the pre-action potential ionic balance across the membrane is achieved by virtue of the action of the Na/K ATPase pump which extrudes sodium whilst accumulating potassium within the cell.

Inhibitory postsynaptic potentials (IPSP's) inhibit by keeping membrane potential from reaching threshold for spike generation. IPSP's achieve this by rapid HYPERPOLARISATION or increases in membrane potential, which reduce the probability that a cell will trigger an action potential.

NERVE CONDUCTION

There are several facts which should be known in relation to nerve conduction - Depolarisation/hypepolarisauon. - Miniature endplate potential(MEPP) - latency. - The speed of propagation is 2-224 mph. - Myelination /non myelination; the former conduct impulses faster than the latter; the myelination on neurones is due to Schwann cells wrapping themselves around th axon. - Saltatory conductance between the Nodes of Ranvier of myelinated neurones is the reason they conduct impulses so rapidly. - Refractory periods (absolute and relative) during the absolute refractory period following an action potential, stimulation will not result in an action potential; during the relatively refractory period, it is difficult to generate an action potential. - Quantal release vesicular hypothesis; there is a large body of evidence, e.g. anatomical and physiological, that neurotransmitters are stored in presynaptic vesicles, that these vesicles fuse with the neuronal plasma membrane during depolarisation and that this results in quantal or "packet-release of the transmitter substance(s). In addition, there is also biochemical evidence that neurotransmitters may be present in the cytosol of the nerve ending and that release can occur from this fraction: in this context, it has also been demonstrated that neurotransmitters are probably present in several discrete intracellular pools and that release from these is not uniform i.e.It may function as long term storage pools.

NEUROCHEMISTRY/NEUROPHARMACOLOGY

UNIQUENESS OF NEURONES

Neurons have an extremely large surface to volume area because of their dendrites and axons. As they are responsive to electrical stimulation and because they use complex, highly organised anterograde and retrograde systems for the fast and slow transport of proteins and transmitters. They are one of the most metabolically active cells in the body. Neurons are unlike other cells in that they have an absolute requirement for glucose and cannot metabolize lipids. In addition, they share the distinction with the lens of the eye, of not requiring insulin in transporting glucose into the cell. Plasticity is another very important specialized property; thereby changes in stimulation will lead to permanent functional changes in the neuron: these could include structural changes in neurone outgrowth or expression of a particular receptor or synthetic enzyme involved in neurotransmitter synthesis. It is reasonable therefore, to conclude that any toxic insult to such a highly active cell with such a large area of membrane, which has increased susceptibility because it has diverse functional parts (eg cell soma, dendrite, synapse), which does not divide, etc, is potentially serious.

CARBOHYDRATES IN BRAIN

The major energy source in brain is glucose and overall glucose metabolism in brain corresponds approximately to that of an unstressed heart: however, local brain activity may increase glucose metabolism substantially. Approximately 20% of body oxygen is used by the brain and 15% of glucose in brain is converted to lactate.

Glucose enters the brain by a carrier mechanism i.e. it is passively transferred across the blood brain barrier (BBB). Glucose deficiency resulting in coma, e.g. due to insulin, will result in irreversible damage after 90 minutes. In terms of recent psychiatric research, the most important aspect of carbohydrate metabolism in brain is the fact that glucose metabolism is proportional to neuronal activity: hence. local glucose metabolism reflects local neuronal firing. This fact has been used to study brain activity in vivo. In such studies, 2-deoxyglucose is used. This is only partially metabolised through the glycolytic pathway and hence this glucose metabolite accumulates. If this 2-deoxyglucose is labelled with a positron emitting isotope with a very short half-life (t 1/2) e.g. 18-fluorine, it can be administered into patients and by scanning their brains using PET, changes in neuronal activity can be observed. It is this procedure which has provided the evidence which has implicated dorsilateral frontal cortex abnormalities in schizophrenia and which has been used in the diagnosis of brain tumours.

LIPIDS

For many years lipids were envisaged as the building "blocks" of brain and other cellular membranes. This is easy to understand on the basis of their distribution and also because of their structure. As can be seen there is a glycerol "backbone" and three long chain fatty acids (in simple lipids). In the case of phospholipids, a phosphate group is present in the third (or first-side chain and in additional inositol phospholipids or phosphatidyicholine have inositol or choline adjacent to the P4.

Most important in the last decade has been the recognition of the fact that phospholipids can be hydrolysed by receptor mediated (e.g. alpha-adrenergic, 5-HT2, some muscarinic, the metabotropic glutainatergic and several peptidergic processes and hence phospholipid metabolites can act as second messengers. The most documented of these is the phosophlipid-system in which the phospholipid phosphatidyl-inositol bisphosphate is hydrolysed I(to give the two intracellular second messengers inositol trisphosphate (IP3) and diacylglycerols (DAG): the main significance of this system is that it appears to be the one which is attenuated by lithium i.e. neuronal Systems involving such receptors will theoretically function with diminished effectiveness.

NEUROTRANSMITTERS

Noradrenaline (norepinephrine) is present in brain as a neurotransmitter in neurones arising from the locus coeruleus (LC). The catecholanime like other monoamines, does not cross the blood brain barrier. It is synthesised from tyrosine and the rate limiting step is at the level of tyrosine hydroxylase. Noradrenaline's effects are terminated by re-uptake into presynaptic terminals. Metabolism is by monomine oxidase type A (MAO A) and by the extracellular enzyme catechol-O­-methyl transferase (COMT). The major metabolites are methoxyhydroxyphenylglycol (MHPG) and vanillymandellic acid (VMA). Both of these (together with various glucuronide and sulphate conjugates are found in urine, blood and CSF but there is not a general consensus as to whether they are significantly changed in psychiatric disorders e.g. as proposed by the Catecholamine Hypothesis of Depression.

5-Hydroxytryptamine (serotonin is an indoleamine and is present in brain in neurones arising mainly from the raphe nucleus. It is formed from L-tryptophan and the rate-limiting step in its synthesis is the availability of tryptophan.

L-trypophan availability is the rate limiting step because its uptake across the BBB requires that it competes with more abundant amino acids. 5HT is metabolised to 5-hydroxyindole-acetic~ acid (5-HIAA) by the mitochondrial enzyme monoamine oxidase type A (MAO A). 5HT is taken up into presynaptic neurones by a specific uptake mechanism which is the site of action of the newer antidepressant drugs such as paroxetine i.e. the so called serotonin specific re-uptake inhibitors (SSRI). The more established antidepressants eg imipramine (Tofranil) and desipramine (Pertofran) inhibit the re-uptake of both 5HT and adrenaline. Alterations in the 5HT system have been implicated in depression but, as is the case with the studies of the noradrenergic system, the urine, blood, CSF and post mortem brain data remain highly equivocal. In the pineal gland, 5HT is the synthetic precursor of melatonin which function remains unclear: it shows a marked increase at night and has been implicated in the control of circadian rhythms and in seasonal breeding. There is pharmaceutical interest in melatonin analogues in relation to jet lag and sleep disorder

Catecholamines, Indoleamines and Affective Illness

The first modern treatment for psychiatric illnesses began in the 1930's with the introduction of ECT: this was based. on the erroneous premise that depression and schizophrenia do not co-exist with epilepsy. Although ECT is still widely used in the treatment of depression its precise mode of action is not yet understood. The next milestone was in 1949 when Cade discovered the antimanic effects of lithium as a result of studies using lithium urate; these studies were examining the effects of urates! Although a great deal of research has been done on lithium, its precise mode of action remains unclear (although its inhibitory effect on inositol-l-phosphatase has produced great interest).

The mood elevating effects of monoamine oxidase inhibitors (MAOI) were discovered in 1956, when Crane noted their effects on patients suffering from tuberculosis and who were being treated with the anti-tubercular drug iproniazid, which is also a MAOI. MAOIs inhibit the action of monoamine oxidase, an enzyme responsible for the breakdown of monoamines. At that point, although the monoamines noradrenaline (NA) (Vogt 1954), and 5-­hydroxytryptamine (5HT) (Erspamer 1953) had been located both peripherally and in the brain, they were not yet established as central neurotransmitters. Reserpine, in use as an antihypertensive, was found to cause some cases of severe depression and as Bertler had shown that reserpine depletes monoamines from tissues, the idea gradually emerged that depleted monoamines might be associated with expressive illnesses.

In 1958, Kuhn reported that imipramine, which is not a MAOI, was an antidepressant. It was shown from studies on blood platelets (Marshall et al. 1960) that imipramine inhibited amine uptake into cells. Amine reuptake into neurones was also suggested to be the primary means ot terminating the effects of monoamines by Axelrod and his colleages (Whitby et al, 1961). This was supported by pioneering work into the synthesis and metabolism of monoamines by Carisson's group in Sweden.

From these and related studies, there emerged the Catecholamine Hypothesis of Depression which proposed that depression was due to a deficit of NA in the brain. The indoleamine 5HT was also proposed to be aetiologically important as it, like N A, has its neuronal reuptake blocked by tricyclic antidepressants. The relative aetiological and therapeutic importance of NA and 5HT has not been resolved though it is clear that they interact in the CNS. However it is interesting to note that much of the recent effort into the development of new antidepressants has been focused towards producing either NA or 5HT specific drugs.

Many of the clinically based laboratory studies on depressive illnesses between 1960 and 1980 were on amines and their metabolites in urine, blood, and cerebrospinal fluid. It was a constant issue as to whether attempts should be made to study amines or their metabolites, study steady states or turnover, and whether to study urine, blood or CSF. Though these studies did not seem to implicate NA rather than 5HT, and though reports of 3-methoxyA-hydroxyphenyl glycol (MHPG) (NA metabolite) and 5-hydroxyindole acetic acid (5HIAA) (5HT metabolite) in depressives have not been proven, it was shown that there is little to be gained from CSF studies (rather than urine or blood) due to the bi­directional flux of many of these substances between blood and CSF.

Ouantitation of receptors using receptor binding techniques were introduced to psychiatry in the 1970's. This move away from amines and metabolites was important clinically because the delay in onset of therapeutic effects of antidepressant drugs could now be explained in terms of the relatively slow changes in receptor populations which occur in response to the presence of agonists or antagonists. The data implicating receptor change in the aetiology of depression and in the effects of antidepressant drugs is derived from both clinical and laboratory studies, although laboratory studies have been hampered by the unavailability of adequate animal models of depressive illness. Thus, most reports are concerned with the long term effects of antidepressant drug administration on normal animals (usually rodents). The most consistent observation is probably the decrease in beta adrenoceptors and adrenoceptor-coupled adenylate cyclase after chronic, but not acute, antidepressant drug administration.

By postulating that antidepressant drugs increase amine levels in the synapse it is reasonable to accept that this will result in a reduction in receptor number and function. However, to integrate such changes into a therapeutic model is not simple. For example the effects produced by antidepressant drugs are similar to those produced by repeated stress, and it has been argued that the changes seen following stress are adaptive and that antidepressant effects are similar. An hypothesis proposing that depression is related to the development of supersensitive beta adrenoceptors, an effect which can be reversed by antidepressant drug administration, was proposed by Sulser et al (1978). However, there does not seem to be a clear explanation of why, for example, the "beta-blockers". which can cross the blood brain barrier are not antidepressants. In a similar way, the aetiology of depression and its treatment has been hypothesised to involve changes in alpha 2-adrenoceptors (Campbell et al. 1987) and 5HT receptors (Gandolfi et al. 1985, for example) but none of these changes have been shown to occur with all antidepressants. The most cynical view with respect to all such studies is that the receptor changes are epiphenomena and predictable on the basis of the pharmacological profiles of the drugs.

Clinical approaches to the problem have led to the development of neuroendocrine challenge tests to assess receptor status in patients. For example an alpha 2-adrenergic agonist, such as clonidine, is administered and the resultant increase in plasma growth hormone (GH) is measured. The major drawback in these types of study is that they assess receptor function in the hypothalamic-pituitary axis and thus, may not be related to changes occurring in parts of the CNS controlling mood.

It has been shown that imipramine causes selective increases in glucocorticoid receptor, immunoreactivity in the locus ceruleus and in 5HT cell groups of the rostral ventromedial medulla. Glucocorticoids are known to act as DNA binding proteins which modify the transcription of specific genes, and have been identified immunocytochemically in the nuclei of 5lHT and catecholaminergic CNS neurones. This, together with the observation that glucocorticoids modulate noradrenergic receptor activity, led Sulser to propose a link between gluconicoids and 5HT and NA in the aetiology of depression. This supports the idea that transcriptional changes may be involved in antidepressant drug action, in this case mediated by a steroid hormone.

Considerable advances are being made in molecular neuroscience and these have significantly changed our perception of the brain. Many neurotransmitter receptors have been isolated and purified and their actions have been classified into three main categories: close coupled receptors (directly linked to an ion channel) such as the nicotinic acetylcholine receptor, those linked by a GTP binding protein to cAMP formation, such as the alpha2, D1 and D2 receptors; and those protein linked to the hydrolysis of inositol phospholipids. It has become clear from receptor purification and structure analysis that receptors exist in families determined by their structural homologies. The receptors 6 protein linked to inositol phospholipid hydrolysis include the following: alpha 1-adrenergic. 5HT2, 5HT1c, muscarinic cholinergic, histaminergic, substance P, bradykinin, and the metabotropic glutamatergic receptor. The major effectors in the process are inositol- 1-triphosphate (1P3), and diacylglycerol (DAG) (Downes, review 1989). These cause changes in intracellular calcium and calcium dependent processes, such as protein kinase activity. The recycling of inositol is necessary for the process to continue. Lithium may produce its therapeutic effect by damping down the CNS phosphatidyl inositol (P1) signalling system via selective inhibition of certain inositol monophosphatases required for inositol recycling (the source of inositol not blocked by lithium, from the blood, is very low in the CNS). There are two main problems with this proposal. One is that the effects of lithium on inositol phosphate accumulation are very rapid in vitro but the clinical effects take days or weeks to appear. The other is that it is quite hard, even in vitro, to demonstrate large reductions in the pool sizes of the inositol phospholipids in the presence of lithium. However these discoveries may lead to the development of non-toxic (possibly organic) inhibitors of the above inositol phosphatases for use in the treatment of manic depressive illnesses.

There are a number of significant questions which remain unanswered in relation to depression and antidepressant drugs. The first is,' do antidepressants cure depression or just ameliorate the symptoms, and do they have prophylactic potential? Are MAOIs therapeutically effective'? Why is there a delay in onset of therapeutic activity for lithium and the antidepressants, when many of the known biochemical effects are relatively rapid? What is the relationship between mania and depression? How does electroconvulsive therapy (ECT) exert its therapeutic action? How can side effects be reduced? It is an awareness of these issues that have led to the proposition that a new approach to the problems would be beneficial. in terms of both the understanding of the aetiology of depression and drug development. The study of gene transcription using molecular techniques is one such new approach.

When appropriate stimuli, either physical, electrical, or chemical, are applied to the brain. Changes in gene transcription occur either to complement or to correct for the effects of the stimulus. There are probably many proteins whose production in the CNS may be affected by neuroactive drug administration. These would include receptors, neurotransmitter synthetic enzymes (tyrosine hydroxylase for example for NA), neuropeptides, ion channels, protein involved in reuptake, brain specific protein kinases, and enzymes involved in the second messenger systems to name a few. Other possibilities might be cytoskeletal proteins and those involved in axonal transport, and proteins involved in the transmitter release mechanism. If it could be found which mRNA levels change, specifically as a result of the presence of an antidepressant drug, an hypothesis based on genomic expression could be proposed leading to the development of more effective drugs.

New approaches are needed to establish the aetiology of depressive illnesses and the mode of action of antidepressants. Although the involvement of monoamines seems apparent there are still many inconsistencies and unanswered questions. By finding out which genes are affected by currently available antidepressants. and as a result which corresponding protein levels are altered, new, more efficacious antidepressants, with possibly lower side effects, could be developed.

L-Dopamine is present in three discrete neuronal pathways, the nigrostriatal the mesolimbic and the tubero-infundibular. The first is damaged in Parkinson's Disease, the second has been implicated in schizophrenia and the third controls (negatively) the release of prolactin (PRL) this latter phenomenon is utilised in a neuroendocrine challenge test of DA receptor function

Dopamine is synthesised from tyrosine and L-Dopa: the latter is administered as a DA precursor in Parkinson's Disease because the monoamine will not cross the BBB.

Peripheral decarboxylase inhibitors are often co administered to prevent L-Dopa being metabolised in the periphery.

Dopamine is metabolised to homovanillic acid (HVA) by monoamine oxidase and this process can be blocked by deprenyl. Dopamine synthesis from L-dopa in the periphery can be blocked by administering peripheral decarboxylase inhibitors such as carbidopa.

There is considerable circumstantial evidence implicating dopamine in the aetiology of schizophrenia, e.g.

1) Antipsychotic drugs are DA receptor antagonists

2) Brain DA receptors may be supersensitive in schizophrenia.

However. there are arguments which can be raised against the validity of some of the conclusions which have been drawn. The most important is the fact that DA receptors can become supersensitive in response to chronic blockade by antipsychotics and hence data from patients especially chronic schizophrenics, may be misleading.

There is general acceptance of the view that central DA systems are involved in reward, e.g. drugs of abuse amphetamine, cocaine, and opiates all increase extracellular level of DA in the nucleus accumbens (a limbic area) whereas aversive agents have the opposite effect

Acetylcholine

This exists in neurones arising from the Nucleus Basalis of Maynert (NBM) and deficits in this pathway have been suggested to have aetiological significance in Alzheimer's Disease.

Acetylcholine is synthesised from choline and acetyl-CoA using the enzyme choline acetyltransferase (ChAT) (This enzyme is used as an immunohistochemical marker to highlight neurones.

Acetylcholine's neuronal release is terminated hy the extracellular enzyme acetylcholine esterase which causes the release of tree choline. This choline is then taken back into neurones by acetyl choline carrier and is re-used in Ach synthesis.

Inhibitors of acetylcholine esterase are used in myasthenia gravis but also (sadly) as nerve gases. Cholinesterase inhibition is also a property of several pesticides.

AMINO ACIDS AS NEUROTRANSMITTERS

For many years there was electrophysiological evidence that amino acids might be neurotransmitters but this idea was treated with some scepticism. However, it is now clear that in fact amino acids are the most universal neurotransmitters in the CNS. They can also have presynaptic effects. Dicarboxylic amino acids such as asparate and glutamate are universally excitatory whereas neutral amino acids such as glycine and g-amino butyric acid (GABA) are universally inhibitory. Other amino acids which are inhibitory alanine, taurine, hypotaurine, pipecolic acid and Gama-amine-N-valeric acid.

The criteria required to establish an amino acid as a neurotransmitter are the same as those which are applied to other substances.

  • A.A. occurs In relatively high concentrations in synaptosomes (or synaptic vesicles).
  • The synthesising enzyme or enzymes are present in synaptosomes or selectively localised in nerve terminals.
  • Calcium-dependent release of A.A. following electrical stimulation of neuronal pathway.
  • Exogenous application of A.A. is physiologically and pharmacologically comparable to action of endogenously released compound.

Secondary or supportive evidence:

  • Selective sodium-dependent reuptake system present
  • Presence of 'receptor' for A.A. demonstrated either by physiological response or binding of labelled ligand.

Amino acids are taken into the brain by several active transport systems which are specific for various groups of amino acids and apart from their obvious role in protein and peptide synthesis. several function as neurotransmitters e.g. glutamate, aspartate, glycine and GABA and several others are precursors of other neurotransmitters e.g. tyrosine and pheylalamine (dopamine, noradrenaline and adrenaline), tryprophon (serotonin) and histidine (histamine).

The transport of amino acids across brain capillaries in vivo falls into three basic groups. one for large neutral amino acids, one for large basic amino acids and one for the group. There is also some differences of amino acids into brain. Transport systems ~ much higher affinity for L than D amino acids.

Glutamic Acid (Glutamate) is a non essential amino acid and the major excitatory neurotransmitter in brain.

Ii has ubiquitous distribution and this together with the fact that it is an amino acid slowed its general acceptance as having a role as a neurotransmitter. Its release in excess leads to excess calcium entry into neurones and hence to neuronal cell death. This process is thought to occur in epilepsy induced damage and in stroke and it is called excitotoxicity. (Glutamate has also been implicated in learning and memory as it is involved in the induction and maintenance of long-term potentiation (LTP).

Gama-Amine-Butyric-Acid (GABA)

GABA is the major inhibitory neurotransmitter in brain. It is synthesised in brain from glutamic acid by the Vitamin B6 dependent enzyme glutamic acid decarboxylase (GAD) which is used as a marker for GABAergic neurones: it is metabolised by GABA transaminase.

It is ubiquitously distributed throughout the brain and has been estimated to be present at 20-40% of synapses. Following its release, GABA is taken up into glial cells where it is decarboxylated to glutamine. The glutamine moves from glia to neurones where it is converted back to glutamic acid.

Minor tranquilisers such as Valium and Librium (ie benzodiazepines) behave electro physiologically similar to GABA, but receptor binding studies have established that they do not act on the GABA receptor. It was also shown that benzodiazepines had no electrophysiological effect in the absence of GABA and that in binding studies, benzodiazepines facilitated the binding of GABA agonists. From these observations, it was established that the compounds such as Valium bind to an accessory site on a GABA receptor complex. However, disinhibition of specific benzodiazepine binding Sites does not always follow those of GABA. Subsequent binding studies established that other compounds could either (a) bind to all same sites as benzodiazepines and yet have no effect on GABA systems (eg electro-physiologically or behaviourally) or (b) hind to ale same site as benzodiazepines and reduce the electro-physiolugical effects of GABA and be anxiogenic. This led to the introduction of all concept of inverse agonist.

Benzodiazepine Binding Sites (Adjacent to GABA receptors)

These benzodiazepine binding sites have been a source of great interest in that many people believe that their existence indicates the existence of an endogenous ligand. However, although some naturally occurring betacarbolines have been shown to bind to the site, their claim to be the endogenous ligand is tenuous, mainly because their effects are only seen at high concentrations. Similarly, numerous adenosine derivatives have been examined as potential endogenous ligands. Decreased numbers of benzodiazepine binding sites ~ been suggested to be a possible factor in epilepsy as genetically selected seizure prone rodents have decreased numbers.

Studies similar to those described above has established that in the GABA receptor, there is also a specific binding site for barbiturates. Like benzodiazepines, Barbiturate facilitate GABA agonist binding and are anxiolytic. They may directly affect .

Chlordiazepoxide (Librium) was the first commercially available benzodiazepine and it was introduced into clinical practice in the l960s. This was followed by diazepam -Valium(1963) lorazepam (Dalmane) and oxazepam (Serax). They have a unique spectrum effects in that they are muscle relaxants. sedatives, anticonvulsants, and have anti-anxiety properties

Tolerance to the sedative and anticonvulsant properties develops but does not develop towards the anti-anxiety properties. They are in widespread clinical use in part because of their high therapeutic index but there is evidence that some dependence can develop and that from behavioural standpoint they may inhibit coping behaviour.

Electrophysiological studies have shown that benzodiazepines act by increasing the frequency of CI-channel opening as opposed to to barbiturates which increase the duration of channel opening and picrotoxin which promotes closure.

Glycine

Glycine is found in highest concentrations in brainstem and in spinal cord where it is located in grey matter. In the spinal cord it is released by interneurons and has a Indirect role as an inhibitory neurotransmitter. The best known glycine receptor antagonist is strychnine. The seizures produced by strychnine and other related alkaloids are probably due to blockade of glycinergic inhibitory neurotransmission at the level of the spinal cord.

Nitric Oxide

In the last few years, it has emerged that EDRF is nitric oxide (NO) and there 1% increasing evidence that this very simple molecule may have a role as a gaseous neurotransmitter in the CNS. The gas is synthesised from arginine via the enzyme nitric oxide synthetase (NOS) which is present in neurones. The gas probably then diffuses out of neurones and into adjacent ones where it activates guanylate cyclase to form the 2nd messenger cyclic GMP. In glutamatergic neuronal systems it appears that the NO is released post synaptically in response to activation of the NMDA type of gutamate receptor : the post synaptically formed NO then diffuses back to regulate the presynaptic neurone ie NO may be a retrograde messenger.

Peptides

The use of immunohistochemical techniques has shown that there are a large number of peptides in brain. In general these act as co transmitters i.e. they are co-released with classical neurotransmitters. For example, substance P and enkephalin may coexist with 5HT, cholecystokinin with DA and VIP with acetylcholine. On the other hand, there are some specific peptidergic neurones, eg for the opiate peptides. Of the several peptide families , the peptides have probably been most extensively examined in the CNS. They are all derived from large prohormones. For example, pro-opiomelanocortin (POMC) is the precursor for alpha and beta MSH, ACTH, and the beta amino acid, beta endorphin. The pentapeptides leu and met enkephalin are processed from proenkephalin and the dynorphins from prodynorphin. In spite of massive amounts of research in this field, it has not really produced much insight into the nature of drug addiction.

NEUROTRANSMITTER RECEPTORS

These exist in various subtypes for each neurotransmitter eg 1 and 2 and BI, and B2 for noradrenaline, SHT (1,2 and 3) DA (1-5) Glutamate (NMDA, Kainate, AMPA) GABA (A and B) acetylcholine (nicotinic and muscarinic) etc.

Receptors in general act in one of 4 discrete ways. They increase or decrease cyclic AMP production (B or alpha 2-adrenergic respectively), they hydrolyse inositol phospliolipids ~ muscarinic. alpha I -adrenergic) or they act directly on ion channels (nicotinic Na+, GABA ICI-1 and glutamate ICI-2). Probably the most important thing about receptors in psychiatric terms is that they are not constant i.e. their number can change in terms depending on input, i.e. denervation/blockade increases receptor number and overstimulation causes a decrease. This has to be considered in the interpretation of clinical data e.g. the reported evidence of super sensitivity in DA receptors in schizophrenia.

Receptors can be measured directly using receptor binding techniques: in essence these measure the specific binding of a radiolabelled ligand for the receptor eg clonidine for alpha-2 receptors or spiropepidol for DA receptors to the tissue either in vivo or in vitro. The two imporant parameters are Bmax( which is a measure of the number of receptors which are present and Kd (which is a measure of the affinity of the ligand for the receptor). The technique is applied clinically in PET scanning studies of receptors: in this case, the amount and distribution of the radioligand in brain is assessed using the scanner. Indirect clinical measures of receptor status: the fact that some receptors control hormone release (e.g. GH is released by alpha-2 adrenergic agonists, PRL release is inhibited by the DA agonist apomorphine, melatonin is released by B-adrenergic agonists) has led to the development and use of neuroendocrine .·challenge'· tests. Measurement of hormones in plasma after a "challenge" by an agonist forms the basis of one form of clinical assessment of receptors. The hormones can be measured using radio-immuno assays (RIA's) using commercially available kits.

THE NEURONAL CYTOSKELETON

This is increasing interest in the intracellular framework which maintains the size, shape and functionality of neurones, because it appears that this cytoskeleton can be disrupted in neurodegenerative diseases. For example, neuronal microtubules are disrupted to form tangles in Alzheimer's disease and the cause may be abnormal metabolism (Phosphorylation) of a microtubule associated protein (MAP) called tan. This leads to the formation of the paired helical filaments (PH F) which form the tangles. Another hypothesis is that a membrane spanning protein amyloid precursor protein (APP) is abnormally cleaved to form B-amyloid which gets deposited in Alzheimer's Disease. The so called abnormal processing of APP is gradually being understood but the problem will not be resolved until the normal function of APP is understood. Whether these various changes are a cause or a result of cell death is unclear. It has been suggested (for ex) that this protein (amyloid) when it is released in excess. causes pores to form in neuronal membranes and that they allow excess calcium into the cell.

I excessive free radical formation due (for ex.) to mutation in superoxide dismutase (SOD) are a failure in calcium homeostasis have also been implicated in neurodegeneration.It is important to recognise that these apparent pathological changes also exist in normal brains.

MOLECULAR BIOLOGY/MOLECULAR GENETICS

During the 60's and 70's DNA/RNA studies were largely restricted to genetics, and oncology and such biochemical investigations came under the broad tide of molecular biology. The availability of the associated technologies in relatively simple kit form has led to a massive expansion in molecular neurobiology.

The basic tenet of molecular biology is DNA -- RNA -- Protein and it is the application of methods for sequencing, comparing, amplifying and modifying these steps that is the basic business of molecular biology.

Genes are made of DNA and these form a double stand of 4 bases, adenine guanine cytosine and thymine. Replication involves the enzyme DNA polymerase. Pieces of DNA that form genes begin with a promoter region and the bits which are expressed are exons and the bits which are not expressed are introns. Genetic information is transformed into proteins via messenger (m) RNA formation (transcription)L: this is catalysed by RNA polymerase. mRNA contains the message for protein formation. Sequence of 3 bases corresponding to an individual amino acid is a codon Protein formation from mRNA (translation) occurs on cytosomes.

Resection enzymes are enzymes isolated from bacteria and which can be used to fragment DNA. The cause breakage at specific points on the DNA and hence the resultant fragments differ between individuals. This is the basis of DNA fingerprinting and restriction length fragment polymorphism (RLFP) which is used in molecular genetics. Southern plots are used to identify DNA fragments: RNA fragments are separated and identified using Northern blots.

Transfection is when a gene is added to a cell. When this is done for whole animals it is transgenics. Of great value to studies of genes, proteins etc. is the ability to amplify genetic material via the polymerase chain reaction (PCR).

CLINICAL PHARMACOLOGY

(MATERIA MEDICA : DRUG TREATM£NTS: GENERAL POINTS

FORMULATION

Pharmaceutical issues: type of preparation, size, colour, dose, packaging, rate of disintegration. Pharmacokinetic issues: absorption, distribution, metabolism and excretion. Pharmacodynamic issues: drug/tissue interactions.

Toxicological Testing: in vivo and in vitro; NOAEL and LOAEL (no observable and lowest observable adverse effect levels); The dose of a drug which will kill 50% of a group of animals i.~ the LDSO and when tested in two species provides an idea on the amounts which ~an be tested in humans. Risk Assessment in relation to the CNS is complicated by the fact that there is no neuronal regeneration and hence toxic effects may eventually be seen in combination with the effects of normal ageing. The therapeutic index (TI) is the ratio of the maximum tolerated dose/minimum effective dose. Obviously a high value is preferred (eg phenothiazines) and if it is low (eg lithium) repeated monitoring of plasma levels is necessary. If f~)ll~)wing testing. a drug with a low TI is found to be clinically valuable but poses the threat of overdose, then prescription guidelines can be made in terms of amounts and other strategies such as individual packaging in foil or the inclusion of a small amount of an emetic in each tablet can be considered. Another important issue in toxicology is to establish whether the new drug will antagonise the effects of noxious substances eg strychnine. In the case of potential antibiotics (say) the drug will be examined in terms of the spectrum of microorganisms it will destroy. However with respect to drugs with potential use in psychiatry, the issue is much more complex. This is because there are limitations on the interpretation of data from animal models of specific psychiatric illness

Following, toxicological and behavioural testing in animals and possibly in tissue cultures, the drug may be introduced into man in a series of stages. The first trial is usually in normal volunteers and data on doses/side effects, and general pharmacokinetics will be determined These studies should also seek to establish whether it is the drug in its initial form or a metabolite which may be therapeutically important. A well documented example of this issue is in the case of imipramine (Tofranil) which is partially metabolised to desmethylimipramine (Pertofran) and for years there was debate on which compound was therapeutically active: it is now established that both are efficacious for the treatment of depression.

A similar issue presently surrounds the MAOI deprenyl (Segiline) which is partially metabolised to amphetamine. Following evaluation of these data, the drug may be approved for a limited trial in hospital patients and subsequently after evaluation, for a series of major trials. Finally it may be approved for hospital use/use by prescription only/general use. Such procedures are under bodies such as the Commission on Safety of Medicines (CSM) (UK) or the Food and Drug Administration (FDA) (USA). Development of a new drug is obviously very expensive with no guarantee of success. However, the existence of 16 year patents should allow recoup of costs.

DRUG TRIALS

Following approval of a drug by a government agency, it is usually necessary for individual hospital ethical committees to examine proposed drug trials. Obviously in the simplest case, the drug is administered to all the sick patients. However evaluation can be difficult in this case and usually there is a comparable untreated group for comparison. This immediately raises an ethical issue ic is it reasonable to withhold treatment from a sick patient. For this reason it is usual to compare a drug with one of known efficacy. In some specific cases. ()~ optical isomer has been tested against the other eg alpha-flupenthixol was tested against beta-flupenthixol: this was because some pharmacological data (eg antagonism of D2 receptors) predicted that the alpha but not the beta isomer would be efficacious as an antipsychotic In the case of psychotropic drugs it is usual now to keep both the patient and the clinical blind to the treatment and have the two drugs or drug and placebo administered a design held in the hospital pharmacy. i.e. this is a double blind trials. It is also common to treat all patients with with drugs eg 6 weeks drug A, 2 weeks washout period, 2 weeks drug B. this is a double blind crossover trials. Such designs are necessary as many psychotropic drugs have limited efficacy and their effects need to be distinguished from those of placebos.

There are numerous other issues which need to be addressed in relation to drug trials. It patients are on drugs. bow long should the "washout" period be before beginning the trial.

How much drug should be given, how often and for how long are also important considerations and some trials address this issue in part by using a design which allows the dose of drug to be increased during the trial. A related issue is the concept of the therapeutic window, i.e. drugs have a level which is optimal and above this can become toxic and below this are ineffective.

There are several other points which the clinician must consider in relation to a trial of a psychotropic drug. Obviously, the choice of patients i.e. sex and age should be considered and correct diagnosis is clinically important as is the means of assessing the patient's psychiatric state before and during the trial. Also, what is the optimum number of subjects necessary to provide statistically meaningful data. Care must be taken to establish it withdrawal of the drug will lead to any dangerous rebound phenomena and a post trial measure of relapse rate should be made. Drug monitoring in plasma should be undertaken to establish if there is a simple correlation with clinical response. This raises the issue of whether the drug is partially bound to proteins or free in plasma and which fraction is therapeutically important (for example. tricylic antidepressants and also chlorpromazine are bound to plasma proteins) Furthermore, the drug may attain steady state levels in plasma or be rapidly cleared. The latter case usually occurs when the drug forms covalent found in tissues (eg as is usually the case with MAOI). Interestingly is has been established that with tricyclic antidepressants. patients on the same dose of drug can show 12O fold variation in plasma levels and furtbermore, that in the case of imipramine at least, plasma level is not a good predictor of therapeutic response.

It is important to establish exclusion criteria from a drug trial eg physical illness, pregnancy etc but also, criteria should be established which require a patient be withdrawn from the trial. Obviously, life threatening changes are accepted but the degree of acceptable side effects should be decided. Such side effects may be temporary (eg at the beginning of the trial) but interestingly in some cases, their presence may be an early indicator of clinical response.

Finally, one should consider whether a drug has a therapeutic effect or merely "floats" the patients until spontaneous remission occurs

ROUTES OF DRUG ADMINISTRATION

1.Intravenous:
This is the most rapid method and also the most quantifiable

2 Subcutaneous: This is quite slow and can involve the use of oil/pellets as depots.

  1. Oral: This is the safest method.

It aIlows self administration and is economical. However, consideration must be given to possible effects of oral enzymes, stomach acid, stomach and gut enzymes, whether the stomach is full or empty and where the drug is eventually absorbed. The frequency of administration of drugs is dependent on their half life (t1/2).

PHARMACOKINETIC ISSUES

It is important to establish the partition coefficient (PC) of a potential drug i.e. how it behaves in a hydrophilic / hydrophobic milieu. This is important because drug absorption or rate of distribution into tissues and (for ex.) across the blood brain barrier is partially dependent on lipid solubility eg, morphine is slowly absorbed into the brain whereas diacetylated morphine (heroin) is rapidly absorbed. In the case of amines, it is unusual for them to be able to cross the blood brain barrier and hence it is usual to administer precursor amino acid - L DOPA is given as a precursor of dopamine. Finally, attempts should be made to establish whether a drug causes enzyme induction which leads to its metabolism. This can in a specific way eg tryptophan induces tryptophan pyrrolase but but also in a non way enzymes such as cytochrome P450 may be induced in the liver: this is essentially important when multi drug schedules are in use.

For most psychotropic drugs. the most important process determining absorption and distribution is passive non-ionic diffusion;

Intravenous :

Rapid and easy to control. The circulation time from the antecubital fossa to the brain is just 15 seconds

Intramuscular:

This may result in highly variable and erratic absorption and the rate ~dfl~ with local blood flow

ORAL:

The majority of psychotropic drugs are weak bases, and therefore are ionised in gastric acid. Most are absorbed in jejunum. Obviously rapid absorption of drugs will occur if they are given on an empty stomach (fast gastric emptying).

DISTRIBUTION

Entry into the brain depends upon the ability of non-ionised drug to pass through capillary basement membranes Diffusion barriers around cerebral capillaries are particularly tight (the "blood-brain barrier")

The brain receives 15% of the cardiac output, but it is only 2% of body weight -therefore it's the most richly perfused organ in the body. The factors affecting CNS penetration are, ionisation, lipid partition coefficient, hydrated molecular size. Plasma protein binding is also a potent variable determining distribution between blood and brain. Only the non-bound fraction is available for diffusion and for most psychotropic drugs more than 90% of total plasma drug concentration is protein bound.

METABOLISM

Some drugs (e.g. lithium) are lipid insoluble and ionised and thus they are excreted directly through the kidneys. The majority of psychotropic drugs are lipophilic and if filtered in this form by the kidney will simply be reabsorbed through the tubules and little net excretion will occur. Conversion to an ionised metabolite is therefore necessary for excretion.

The primary site of drug metabolism is the liver and oxidation/conjugation are the commonest enzymatic processes. The majority of an ingested dose may be metabolised by enzymes in the intestinal mucosa and liver before reaching the systematic circulation: this is known as "first-pass metabolism". For some drugs, such as pheneline, there is evidence for genetically inherited differences in metabolic ability.

Half-Life:

Time during which plasma drug concentration falls to half its initial value. It usually requires approximately five half-lives to reach "steady state" when starting medication with standard repeat doses, and a similar time for effective washout. For drugs with a long half life it may be sensible to start with a large initial loading dose, if side-effects will be tolerable.

Pharmacological Half-Life: Time taken for the total amount of drug in the body to decline by one halt

Half-Life:

Time taken for the pharmacological effect to fall to half of its initial value

Steady State :

Where amount of drug entering the body equals the amount lost by elimination in a 24 hour period. Theoretical rather than real; although net effect is zero, fluctuation in drug concentration still occur.

Maintained Dosage Concentration:

A more realistic term than "Steady State". Gives the drug concentration at a specified time after the last dose, once the drug has been given for sufficient time to allow the best approximation to the theoretical "steady state".

Dosing Interval: ###

Frequency of drug administration required to maintain plasma drug concentrations within specified tolerance limits about the desired therapeutic concentration.

Dose interval Fluctuation tolerance (expressed as a fraction)

It is rarely necessary to give a drug more frequently than 70% of its half-life, but this will depend Upon the degree of acceptable "peak and trough" levels/effects.

Where an optimal range of drug concentrations exists, below which and above which therapeutic effect is either lost or complicated by toxicity.

^Plasma Half-Lives of some commonly used drugs^t1/2 Hours| |Amitriptyline| 24-48| |Imipramine| |Lithium|8-12| |Chlorpromazine |20| |Benzodiazepines: Diazepam, Chlordiazepoxide |> 30| |Long-acting: Medazepam, Clorazepate, Nitrazepam,Oxazepam
|Short-acting: Temazepam,Lorazepam|< 30|

PROBLLMS ARISING AS A RESULT OF THE BLOOD BRAIN BARRIER.

The capillary endothelial cells of the brain that constitute the blood-brain barrier (BBB) severely restrict the movement of non-transported, polar molecules from the capillary lumen to the extracellular fluid of the brain. The BBB's ability to precisely control the passage of molecules is a function of specialized characteristics such as tight junctions, a paucity of vesicular activity and lack of fenestrations and transcapillary channels. The blood­rctinal barrier (BRB), however, consists of two morphologically distinct sites: the retinal capiIlaries. which are identical to those in the brain and the retinal pigment epithelial cells which are also joined by tight junctions. The function of both the BBB and BRB are essentially identical and factors that influence one are also likely to affect the other.

It is accepted that a large proportion of neurological and ophthalmological problems could be better addressed if viable strategies existed for the delivery of polar drugs into the CNS. The importance of developing such regimes cannot be underestimated.