Intoduction
The term limbic system is applied to a collection of brain structures found in all mammals which is involved with a variety of functions including epinephrine flow, emotion, behavior, motivation, long-term memory, and olfaction. Emotional life is largely housed in the limbic system, and it has a great deal to do with the formation of memories. The limbic system has great input on thirst, hunger, sexual behavior, and reward. Many drugs of abuse such as cocaine and amphetamines directly affect the structures of the limbic system. Perhaps the simplest way to understand the functions of the limbic system is to use the mnemonic “M-O-V-E.”
M – Motivation and Memory formation
O – Olfaction or sense of smell
V – Visceral of autonomic nervous system functions
E – Emotional components of behaviour
All mammals have a limbic system, however many specie variants developed related to the magnitude olfaction plays in the animal’s evolution. In species where olfaction development and function is high (canines, rodents, etc.), the limbic system constitutes more of the brain mass and less devotion to outer cortex development (Finlay et al., 2001). Primates, insectivores, and ungulates collectively demonstrate an inverse relationship between cerebral cortex and limbic volumes, but terrestrial carnivores have high relative volumes of both; meanwhile bats have low relative volumes of both (Reep et al., 2007). Marine mammals with reduced olfactory bulbs also have a reduced limbic system overall (Clark et al., 2001).
Structures of the Limbic System
The Limbic System consists of cortical and subcortical components that have developed from the telencephalon, diencephalon and mesencephalon. The cortical part comprises interconnected telencephalic structures on the medial and basal aspect of the hemispheres, namely the cingulated gyrus, hippocampus and the piriform lobe. The subcortical part includes components of the diencephalon (habenula, hypothalamus, thalamus), midbrain (tegmental nuclei) and the amygdala. The limbic system receives olfactory input from the amygdala and piriform lobe that can trigger emotional behavior such as fear, aggression, and apparent pleasure.
Sheep Brain: Image of sheep brain was taken in 2008 at St. Mary’s College of Maryland under the guidance of Dr. Anne Marie Brady in a Biological Psychology course.
Dog Brain Extraction
The extraction, fixation, and dissection of the dog brain was completed at Szent Istvan University under the guidance of Dr. Kalman Czeibert. Skin surround skull was incised, then using scalpel, the temporal and masseter muscles were transected and scrapes off skull. Using a surgical saw, a rostral cut was made into the frontal sinus. Superficial cuts were made laterally on the temporal bone starting at the occipital condyles, through the nuchal crest and along the temporal bones connecting to the rostral cut at the frontal sinus. Using a surgical chisel and hammer, the superficial cuts were widened allowing the skull to easily be removed dorsally. Carefully, the cranial nerves were removed and the brain was extracted from the cranial cavity.
Fixation
The structure and consistency of the dog brain upon removal was not ideal for dissecting in order to expose the limbic system. Upon removal of the canine brain, it was placed in a 7% formaldehyde solution for 24 hours than transferred into a 3% formaldehyde solution for another 24 hours.
Dissection
The two hemispheres were gently widened, exposing the white matter of the corpus callosum. A light incision was made on the corpus callosum (Figure 1), removing the dense axon fibers connecting the two hemispheres, and making visible the third ventricle and choroid plexus (Figure 2). Using the borders of the lateral ventricles, gently a transverse incision was made through the parietal lobe as well as a lateral cut that would continue all the way through the occipital lobe (Figure 3). The horn of the hippocampus was made visible. Using finite and precise cuts, the remainder of the cortex was removed around the hippocampus (Figure 4). Through a similar process, the remaining part of the left hemisphere was removed, making the thalamus and other components of the limbic system visible (Figure 5&6).
- A second brain was similarly fixed and used for cross sectional dissections. Approximately 5-9 mm thick cuts were made transecting through the canine brain from a dorsal to ventral position. Brain slices allowed the exposure of the internal components of the limbic system (Figure 7).
The various components of the limbic system are each associated with different functions. The Amygdala is involved with several emotional responses (Sato et al., 2016); the Hippocampus is involved with learning and memory formation (Mao et al., 2015); the Thalamus relays sensory and motor signals plus regulates sleep and alertness (Li et al., 2014); the Nucleus Accumbens with reward, pleasure and addiction (West et al., 2016); Hypothalamus is involved with regulation of the pituitary gland and hormonal function (Perez et al., 2016); Olfactory bulbs involved in the perception of smell and connect to piriform lobe & amygdala (De La Rosa et al., 2015); the Habenula receives input from the limbic system and basal ganglia, influencing the brains response to pain, stress, anxiety, sleep and reward (Velasquez et al., 2014).
Molecular and Cellular Neuroscience
- Neuroscience is an expansive field of science which integrates multiple fields of research and investigation in order to understand the functioning of this complex system. The anatomy and physiology of the mammalian brain neural systems have been thoroughly investigated and documented; yet, new discoveries are still being unveiled every day. Through immunohistological studies of many animal species, the location and functioning of neurons and glia (the cells that compose the central and peripheral nervous system) have been identified. Generally speaking, the neuron is the implementation system of the nervous system and the variation of their composition generates the many capabilities possible. The structure of a neuron consists of three components:
Soma – cell body - location of typical cell functions and genetic information storage
Axon - a very slender nerve fiber - projects from the neuron's cell body and transmits electrical impulses to the neuron's axon terminals. This electrical signal stimulates release of a neurotransmitter, which is produced inside the nerve cell. It is neurotransmitters that light up the brain with life.
Dendrites – multiple short branched extensions of a neuron, along which impulses and neurochemical communication is received from other cells at synapses are transmitted to soma.
Neurotransmission
As an action potential travels down the axon, the depolarized cell membrane opens voltage gated calcium channels at the terminal button and stimulates vesicular release. These synaptic vesicles are filled with a particular neurotransmitter (neuro-chemical). There are over 100 different neurotransmitters that have identified in the mammalian nervous system. The location, post-synaptic effect, metabolism, and removal of these neurotransmitters significantly differ from one another. Neurotransmitter transport systems are integral to the release, re-uptake and recycling of neurotransmitters at synapses. High affinity transport proteins found in the plasma membrane of presynaptic nerve terminals and glial cells are responsible for the removal from the extracellular space of released-transmitters, thereby terminating their action. In addition to these removal systems, several neurotransmitter-specific enzymes are present to inactivate or regulate these chemicals released into the synapse. Upon bonding to a post-synaptic receptor, these neurochemicals will typically have an excitatory or inhibitory response on the sequential neuron through ionotropic or metabotropic functions. Through electrophysiological studies, immunohistochemistry, and genetic knock-out models, the understanding of these brain chemicals has vastly improved in the past couple decades. In regards to the topic of this essay and its relation to the Limbic System, we are only going to focus on the neurotransmitters specifically relevant.
Neurotransmitters And Corresponding Receptors
Catecholamines
Classification of neurotransmitters based on their organic structure. Dopamine (DA), Norepinephrine (NE), and Epinephrine (E) belong to this class based on the fact that they are all variants of the organic diphenol, Catechol, as well as containing an amine group. These biogenic amines are derived from the amino acid tyrosine. Tyrosine is created from phenylalanine via hydroxylation by the enzyme phenylalanine hydroxylase or is ingested directly from dietary protein. It is then sent to catecholamine-secreting neurons where many kinds of reactions convert it to dopamine, to norepinephrine, and eventually to epinephrine depending on the neuron type. Dopamine and Norepinephrine have strong implications in the Limbic System and will be described in detail.
Dopamine
This neurotransmitter has many effects systemically, but in relation to its function in the central nervous system, Dopamine has been connected to the involvement in the reward circuitry of the brain as well as control of locomotion, cognition, endocrine function and feelings of engagement or excitement (Vokow et al, 1998; Shen, 2008).
Dopamine Synthesis
Dopamine is synthesized in cell groups in the midbrain's Substantia Nigrae, Ventral Tegmental Area (VTA) and Hypothalamus (Zhaoliang, 2004).
Dopamine Receptors
There are five sub-classes of Dopamine receptors scattered throughout the body of all mammalian species and they are all members of the Rhodopsin like G-Protein Receptor family (7-helix trans-membrane proteins). All of these receptors are metabotropic and involve a second messenger G-Protein (Kienast et al., 2006). Dopamine receptors are typically classified as either a D1 or D2 class, depending on their ability to either inhibit or activate adenylate cyclase and a cascade of events in the post-synaptic neuron (Le Crom et al., 2003).
Class D1
Stimulate G-Proteins and activate adenylate cyclase. The action of the enzyme causes the conversion of adenosine triphosphate to cyclic adenosine monophosphate (cAMP) (Hussain et al., 2003).
D1A - (D1) – Found to have the greatest concentration inside of the nucleus accumbens, caudate putamen, olfactory tubercle; with lesser concentrations found in the pre-frontal cortex, amygdala, hypothalamus, thalamus, and basal ganglia (Hummel et al., 2002). Peripherally they are found in the kidney, heart, parathyroid, smooth muscle and juxtaglomerular apparatus of the renal tubules. D1A are the most common stimulatory dopamine receptor. (Missale et al., 1998; Centoze et al., 2005).
D1B - (D5) – Found to have the greatest concentration inside of the nucleus accumbens, caudate putamen, and thalamus (pain involvement); with lesser concentrations found in the pre-frontal cortex, habenula, amygdala, and hypothalamus. Peripherally they are found in the kidney, heart, liver, parathyroid, and smooth muscle (Missale et al., 1998; Centonze et al., 2005).
Class D2
Coupled to Inhibitory G-Proteins, this class inactivates adenylate cyclase, inhibits phosphatdylinositol, activates K+ Channels and inhibits Ca2+ (Le Crom et al., 2005).
D2- Found to have the greatest concentrations in the nucleus accumbens, striatum and basal ganglia. They are located on the cell bodies of neurons in the ventral tegmetal area (VTA) and substantia nigra. Peripherally they are found in the pituitary gland, heart and blood vessels. Most common inhibitory dopamine receptor. (Murray et al., 1994)
D3- Found to have the greatest concentration in the limbic area, nucleus accumbens, and hypothalamus, with lesser concentrations found in the caudate putamen, cortex and cell body of substantia nigra. May have a role in cognition, emotion and addiction. (Murray et al., 1994; Mugnaini et al., 2013)
D4- Found to have the greatest concentration in the medulla, amygdala, hippocampus, midbrain and prefrontal cortex, with a lesser concentration in the striatum (Rubinstein et al., 1997).
Dopamine Metabolism
As dopamine is released into the synapse, there are many mechanisms designed to remove or inactivate this chemical following neurotransmission.
Dopmanine Transporter
The dopamine transporter is critical for the removal of dopamine from the extracellular space, following its release, and is the principal site of action for psycho-stimulant drugs, such as cocaine and amphetamines, which inhibit the transporter's activity (Giros et al., 1992). A single form of dopamine transporter has been isolated from humans and other mammals. Targeted gene disruption of the dopamine transporter has confirmed its importance in maintaining low extracellular dopamine levels (Gainetdinov et al., 1999).
Monoamine Oxidase
Attached to the mitochondria, this family of enzymes catalyzes the oxidation of monoamines through deamination including serotonin, dopamine, norepinephrine and epinephrine. They are found bound to the outer membrane of mitochondria in most cell types in the body (Kumagae, 1991).
Catachol-O-Methyl-Transferase
Found membrane bound inside of neurons in nearby glia, this enzyme catalyzes the transfer of a methyl group from S-adenosylmethionine to catecholamines, including the neurotransmitters dopamine, epinephrine, and norepinephrine. (Lachman et al., 1996). This O-methylation results in one of the major degradative pathways of the catecholamine transmitters. It is involved in the inactivation of dopamine in brain regions in which the dopamine transporter (DAT1) is sparsely expressed (Jugurnauth et al., 2012).
Trace Amine Associated Receptor
A recently discovered endogenous receptor that stores amine metabolites and monoamines that is also a member of the rhodopsin-like G-protein coupled receptor (GPCR) family. (Miller, 2011) Since trace amine associated receptors are putative endogenous receptors for trace amines. believed to be a key regulator of common and trace brain monoamines (Lindemann et al., 2008)
Veiscular Monoamine Transporter
a transport protein integrated into the membrane of synaptic vesicles of presynaptic neurons.(Yulung et al., 2015). It acts to transport monoamine neurotransmitters – such as dopamine, serotonin, norepinephrine, epinephrine, and histamine – into the vesicles, which release the neurotransmitters into synapses as chemical messages to postsynaptic neurons (Wang et al., 2016; Freyberg, 2016)
Neural Pathways
Brain regions such as the substantia nigra (SN) and ventral tegmental area (VTA) are known to contain dopaminergic cell bodies (Giros et al., 1999). These regions have projects to other locations in the brain where dopamine is released at the axon terminal
Mesolimbic Pathway
Dopaminergic tracts originate from the ventral tegmental area and synapse with the nucleus accumbens. This pathway is known as the reward circuit and is activated during pleasurable stimuli such as food, success, sex, and psychoactive drugs (Joseph et al., 2016). Drug addiction has been associated to this pathway.
Mesocortical Pathway
Dopamine releasing pathway that connects the ventral tegmental area (VTA) with the prefrontal frontal cortex (PFC). It is essential for normal cognitive functioning and is involved in cognition, motivation and emotional response. The D1 class of receptors are more common found in the cerebral cortex (Puig et al., 2014).
Nigrostriatal Pathway
Although irrelevant to the functioning of the limbic system, some of the side effects of drugs that effect the physiology of dopamine will also impair this pathway. The nigrostriatal pathway in locomotion and motor activity. D1A and D2 Receptors are in high concentration inside this system (Song et al., 2016).
Tuberoinfundibular Pathway
A recently discovered pathway of the nervous system. Dopamine producing neurons inside of hypothalamic arcuate nucleus have projections to the pituitary gland. Acting as a feeback control system, the hormone prolactin is tonically inhibited by dopamine secretion. Implications for this system may involve the control of lactation, sexual libido, fertility, and body weight (Ramirez et al., 2015).
Norepinephrine
Norepinephrine has multiple roles within the central nervous system and the periphery. First, it relays messages in the sympathetic nervous system, as part of the autonomic nervous system's fight-or-flight response. Secondly, norepinephrine prepares the brain to encounter and respond to stimuli from the environment, thereby facilitating vigilance. So in both roles, norepinephrine mediates arousal (Zaniewska et al., 2015).
Norepinephrine Synthesis
Neurons in the loci coerulei, a pair of structures located within the pons of the brain stem synthesize norepinephrine (Aston-Jones et al., 2016). Dopamine is further oxidized by dopamine beta hydroxylase to create norepinephrine (Kim et al., 2002).
Receptors
There are three-classes of norepinephrine adrenoreceptors scattered throughout the body of all mammalian species and they are all members of the Rhodopsin like G-Protein Receptor family (7-helix trans-membrane proteins). All of these receptors are metabotropic and involve a second messenger G-Protein (Shen et al.; 2008). The three classes are alpha 1- α1 (a Gq coupled receptor- postsynaptically excitatory), alpha 2- α2 (a Gi coupled receptor- presynaptically inhibitory of noradrenaline and beta ᵝ (a Gs coupled receptor), and each can be further divided into subtypes (Ma et al; 2004).
Alpha 1A
Highest levels being found in the olfactory system, hypothalamic nuclei and in regions of the brainstem and spinal cord related to motor function (Day et al., 1997). The receptor also found in the liver, prostate and urethra and the human heart (Nicholas et al., 1991; Acosta et al., 1999)
Alpha 1B
High levels occurring in the CNS, mainly in the cerebral cortex and brainstem (Acosta-Martinez et al., 1999), and in peripheral tissues such as the kidney and lung (Day et al., 1997)
Alpha 1D
High levels in peripheral tissues, such as the vas deferens and in the CNS, mainly the hippocampus, cerebral cortex and brainstem (Nicholas et al., 1991; Day et al., 1997).
- Alpha 2A
Expressed at high levels in the CNS, and in peripheral tissues such as kidney, aorta, skeletal muscle, spleen and lung (Blaxall et al., 1994; Eason et al., 1997)
Alpha 2B
Found in the kidney, brain, and spinal cord, along with the other alpha 2 subtypes. However, it is the only subtype to be found in the heart and liver. (Eason et al., 1997) Peripheral tissues, predominantly express the alpha 2A and 2B subtypes, with little alpha 2C. This is in contrast to the CNS, where alpha 2A and 2C are predominantly expressed, with little alpha 2B (Blaxall et al., 1994)
Alpha 2C
Receptors found mainly in the brain and kidney, and is absent in spleen, aorta, heart, liver, lung, skeletal muscle (Eason et al., 1997).
1, 2 & 3
Receptors are predominantly found in the periphery and limited relevance with limbic
Metabolism
Since Norepinephrine is also a monoamine and catecholamine, similar metabolism occurs that were previously reviewed with dopamine.
Plasma Membrane Monoamine Transporter
General transporter located on the presynaptic neuron for all monoamines (Serotonin, Norepinephrine, Epinephrine, and Dopamine). It is critical for the removal of epinephrine from the extracellular space (Naganuma, 2014).
Monoamine OxidaseCatechol-O-Methyl-Transferase
Attached to the mitochondria, this family of enzymes catalyzes the oxidation of monoamines through deamination including serotonin, dopamine, norepinephrine and epinephrine. They are found bound to the outer membrane of mitochondria in most cell types in the body (Kumagae et al., 1991).
Catechol-O-Methyl-Transferase
Attached to the mitochondria, this family of enzymes catalyzes the oxidation of monoamines through deamination including serotonin, dopamine, norepinephrine and epinephrine. They are found bound to the outer membrane of mitochondria in most cell types in the body (Kumagae et al., 1991).
Trace Amine Associated Receptor
A recently discovered endogenous receptor that stores amine metabolites and monoamines that is also a member of the rhodopsin-like G-protein coupled receptor (GPCR) family. (Miller, 2011) Since trace amine associated receptors are putative endogenous receptors for trace amines. believed to be a key regulator of common and trace brain monoamines (Lindemann et al., 2008)
Veiscular Monoamine Transporter
- A transport protein integrated into the membrane of synaptic vesicles of presynaptic neurons.(Yulung et al., 2015). It acts to transport monoamine neurotransmitters – such as dopamine, serotonin, norepinephrine, epinephrine, and histamine – into the vesicles, which release the neurotransmitters into synapses as chemical messages to postsynaptic neurons (Wang et al., 2016; Freyberg et al., 2016)
Neural Pathways
The axons of neurons in the loci coerulei project to both sides of the brain where they innervate and release norepinephrine in wide ranging areas. Branching axons of norepinephrine-producing neurons in the loci coerulei innervate the brain stem, spinal cord, and cerebellum, as well as the hypothalami, thalamic relay nuclei, amygdalae, and neocortex (Aston-Jones et al., 2016). Norepinephrine which is most associated with learning and attention problems in our modern-day life (Zaniewska et al., 2015). Selective depletion of NE in the forebrain makes animals more distractible. It is the neurotransmitters norepinephrine and dopamine that accomplish attentiveness in the prefrontal cortex (Golirzaei, 2016).
Monoamines
Monoamines are general description of neurotransmitters that are all derived from the amino acids phenylalanine, tryptophan or tyrosine. As previously described, the catecholamines (dopamine, norepinephrine and epinephrine) are specific class of monoamines. Similarly, histamine, melatonin and serotonin are classes of monoamines due to their derivation from one of these aromatic amino acids.
Serotonin (5-HT)
From a broad perspective, whereas it is dopamine that primarily drives the seeking and reward system, it is both norepinephrine and dopamine that drive the vigilance system, while serotonin acts as a modulator of these neurotransmitters. The transmission of serotonin has a strong influence on the transmission of these and other neurotransmitters. Serotonergic neurons play a fundamental role in the integration of behavior in the central nervous system, but it also holds large roles in the gastrointestinal tract and blood platelets (Nicholas et al., 2008). Our sense of well-being and our capacity to organize our lives and to relate to others depend profoundly on the functional integrity of the serotonergic system. Many distinct emotions share generalized components such as acetylcholine, norepinephrine, and serotonin systems for the control of attention and general arousal functions. There are only a few hundred thousand serotonergic neurons in the human brain, roughly one millionth of the total population of neurons in the human central nervous system (Burnet et al., 1995).
Synthesis
Serotonin is synthesized from the amino acid tryptophan and must be obtained from dietary sources; deprivation alters brain chemistry and mood. Tryptophan is obtained by the digestion of proteins and is abundant in meats. Once digested in the gut it is transported in the blood plasma to the brain, where it is converted to serotonin.
Receptors
Fourteen types of serotonin receptors have been discovered so far in the brains of mammals, located in different places and acting in different ways Due to the magnitude of serotonin receptors located throughout the body, this project will only focus on the receptors relevant to the limbic system and amphetamines. Receptors for 5-HT mediate both excitatory and inhibitory neurotransmission, and modulate the release of many neurotransmitters including glutamate, GABA, dopamine, epinephrine/norepinephrine, and acetylcholine, as well as many hormones, including oxytocin, prolactin, vasopressin and cortisol (Nicholas et al., 2008). In the CNS, 5-HT receptors can influence various neurological processes, such as aggression, anxiety and appetite and, as a, result are the target of a variety of pharmaceutical drugs, including many antidepressants, antipsychotics and anorectics (Aznar et al., 2003). With the exception of the 5-HT3 receptor, which is a ligand-gated ion channel, all 5-HT receptors are members of the rhodopsin-like G protein-coupled receptor family and they activate an intracellular second messenger cascade to produce their responses (Le Crom, 2005).
5-HT1A
5-HT1A receptors are the most widespread of all the 5-HT receptors and are particularly high density in the limbic system (Aznar et al., 2003). They are found pre- and post-synaptically, (Burnet et al., 1995) in the raphe nuclei they are somatodendritic and act as autoreceptors to inhibit cell firing; whereas postsynaptic 5-HT1A receptors are present in a number of limbic structures, particularly the hippocampus (Aznar et al., 2003). 5-HT1A receptors are involved in many neuromodulative processes, and are potential anxiolytic and hypertensive targets. In the CNS, 5-HT1A receptors exist in the cerebral cortex, hippocampus, septum, amygdala, and in the raphe nucleus in high densities, whilst lower amounts also exist in the basal ganglia and thalamus (Burnet et al., 1995).
5-HT2A
5-HT2A receptors are one of the main excitatory serotonin receptors and are expressed throughout the central nervous system in high concentrations found on the apical dendrites of hippocampal pyramidal cells (Aznar et al., 2003). These receptors regulate dopamine release as well as modulate cognitive processes by enhancing glutamate release in the cerebral cortex (Burnet et al., 1995).
5-HT2C
5-HT2C receptor distribution is limited to the central nervous system and the choroid plexus (Nicholas et al., 2008). Activation of the receptor has been shown to exert an inhibitory influence upon frontal cortical and striatal dopamine and NE (Milan et al., 1998)
5-HT6
5-HT6 receptors decrease monoamines in the prefrontal cortex by stimulating; antagonism of these receptors facilitates dopamine and norepinephrine release in the frontal cortex (Lacroix et al., 2004).
Metabolism
Serotonin Transporter
The serotonin transporter is critical for the removal of serotonin from the extracellular space, following its release, and is an active site of action for antidepressants (Giros et al., 1992). Targeted gene disruption of the transporter has confirmed its importance in maintaining positive emotions (Gainetdinov et al., 1999).
Monoamine Oxidase
Vesicular Monoamine
TransporterNeuralPathways
Other Relevent Neurotransmiters
Glutamate
Behavioural Effects of Amphetamines
Amphetamine Effect on Neurotransmitters
Dopmanine Transporter Monoamine Oxidase Inhibitor VMAT Malfunction Catechol-O-Methyl-Transferase
Glutamate Effect
Dependance and Withdrawl
References
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