Two horizontal forces, 225 N and 165 N, are exerted on a canoe. If these forces are applied in the same direction, find the net horizontal force on the canoe.
Final Exam Organization of the Brain and Behavior - In the 16th and 17th centuries there was an explosion of theories regarding the basic regional or topographic organization of the CNS after the dual brain account of Aristotle was introduced in classical antiquity (about 340 BC). Two new classes of theory, evolutionary and genomic, were added in the 20th century; the former has had little substantial inﬂuence, whereas the latter is maturing. - Segmental models of brain organization: essentially consist of a trunk generating regularly spaced paired nerves, and suprasegmental cerebrum and cerebellum • Haller reﬁned the Vesalian model in the 18th century; he was the ﬁrst to distinguish between the medulla and pons • Meynert published a anatomical analyses based on the Vesalian model in 1872; ﬁrst to divide the traditional posterior cerebral ganglion into a dorsal zone (thalamus) and a ventral zone (hypothalamus) Varolio’s basic model has remained popular today; reﬁned by divisions of the • central trunk into a brainstorm part, and generating cranial nerves and a spinal cord part, generating spinal nerves - Developmental models of brain organization: • Baer provided simple, descriptive names for the embryonic brain vesicles ﬁrst identiﬁed by Malpighi; he recognized the forebrain, midbrain, and hindbrain • Ahlborn proposed that the hindbrain developed adjacent to the notochord and is thus epichordal, whereas the forebrain and midbrain develop rostral to the notochord and are thus prechordal - Evolutionary model of brain organization: • Edinger proposed a distinction between the old brain and new brain; he said the old brain is common to vertebrates and is responsible for all sensory-motor reﬂexes and instinctive behavior - Genomic model of brain organization: • The ultimate goal of the genomic model is to decipher and understand the genetic program that assembles the neural tube by analyzing spatiotemporal patterns of gene expression through development and establishing their functional signiﬁcance Central Nervous System Organization: I. Encephalon (brain) A. Prosencephalon (forebrain) 1. Telencephalon (endbrain: limbic system, basal ganglia, cerebral cortex) Diencephalon (interbrain: hypothalamus, thalamus) 2. B. Mesencephalon (midbrain: tectum, tegmentum) C. Rhombencephalon (hindbrain) 1. Metencephalon (pons, cerebellum) 2. Myelencephalon (medulla oblongata) II. Medulla Spinalis (spinal cord) - The brain uses hierarchical organization; a hierarchy is ordering along a dimensions or arrangement in a graded series - The central nervous system uses hierarchical sensory processing and motor control - Amphioxus: a primitive vertebrae with a neural tube as a nervous system; it has no eyes and uses photosynthetic skin cells to detect light - Encephalization is the increase in the size of the rostral area of an organism; the amount of brain mass related to an animal’s total body mass - Encephalization Quotient (EQ): the proportion of an organisms head size to its body; believed to reﬂect intelligence Hierarchical Control by the CNS: - Spinal Cord basic sensory reception, integration, motor command; withdrawal reﬂex • central pattern generators are primitive circuits that involve no sensory input; they • generate rhythms like swimming, respiration, chewing, and walking; the rhythms created by central pattern generators are modulated by reﬂex arcs - Brainstem • cranial reﬂexes (central pattern generators: respiration, mastication) • command systems • primitive responses (orienting, posture, eye movements) • background pattern for more differentiated responses (arousal) • autonomic responses (cranial nerve 10) - Subcortical Forebrain • thalamus: crude discrimination such as light-dark; attention • limbic system: gives rise to speciﬁc emotional states; does not involve physiological stress • hypothalamus: responsive to internal chemistry (homeostasis, hormonal regulation); gives commands as result of deviations from homeostasis (water balance, temperature) - Cortex • ﬁnest sensory discrimination and motor coordination highest integrative activity: cognition, curiosity, consciousness • - “Ontogeny recapitulates phylogeny”: the evolutionary history of an organism is repeated in its development - Behavior can also be organized hierarchically; this reﬂect the order of control over behaviors - Reﬂexes: predictable, stereotyped responses to speciﬁc stimuli • usually localized • involuntary involve relatively few neurons • • graded responses (vary in intensity) • degree of voluntary control over reﬂex varies (holding your breath) • micturition reﬂex: controlling urination is well-controlled; pupillary reﬂex: dilation of pupils are generally not subject to signiﬁcant voluntary control - Rhythmic Behaviors • basis of rhythmic behaviors are central pattern generators (CPGs) in brainstem and spinal cord • mechanisms can operate without any sensory input • • run a sequence of movements • input may be necessary to initiate them - Kineses: change the rate of movement • simple change in rate of rhythm; may be positive or negative to increase or decrease speed of movement photokineses: changing rate of movement as a result of change in amount of light; • thermokineses: changing rate of movement as a result of change in amount of heat • serve to maximize time in favorable environments and minimize time in unfavorable environments • sometimes mistaken as higher order processes: ants move toward cold, dark, moist places, so they are usually seen in groups; the ants appear to be social animals - Taxes: movement oriented by a stimulus • may result in movement toward or away from stimulus depending on mechanism and type of stimulus; chemotaxis: moving toward a chemical, possibly to ﬁnd food - klinotaxis: single receptor integrating over time; “temporal summation”; seen in olfactory sense - tropotaxis: paired receptors working simultaneously; “spatial summation”; such as using two ears to decipher which direction a sound is coming from; moths use tropotaxis to keep the amount of light shining on either wing equal so that they remain balanced - telotaxis: orientation toward a goal; not necessarily related to the stimulus strength; such as swimming to shore - Species- typical behaviors (“instinctive”): behaviors universal to a species but that are not necessarily innate; they may involve minimal learning • AKA ﬁxed action patterns • relatively complex behaviors that are common to members of a species • birds make sounds before they learn the adult song; human babies babbling before learning language - Motivated behaviors (“operant”) • “operant”: learned through reward and punishment • if the behavior is not affected by rewards or punishments, it is instinctive • goal directed • elicited by physiologically meaningful stimuli; deviation from homeostasis • signal food, sex, water, threat, etc • terminated by consummatory response: eating, drinking, ﬁght, ﬂee, copulate • appetitive: approach to stimulus (reward); aversive: withdraw from stimulus (punishment) - Latent Learning • observation, modeling, imitation • my performing without reward • by exposure • learn associations between stimuli (stimuli that do not punish or reward) • associated with mirror neurons exposure: rats learn a maze by exploring • - The hierarchical order of behaviors moves from caudal to rostral as they become more complex (reﬂexes to latent learning); this implies that more encephalized animals have relatively richer repertoires of complex behaviors than do less encephalized animals Development of the CNS - Gastrulation occurs during the third week of gestation in humans - Gastrulation occurs in mammals when the three germ layers differentiate - Migration of mesoderm under ectoderm induces ectoderm to become neural tissue; deﬁnes anterior/ posterior directions (head and tail end) - Three germ layers: ectoderm: neural plate and epidermis; neural plate becomes notochord which • eventually becomes the spinal chord; the neural tube closes from the middle of the chord and rostrally to the brain and caudally to the end of the spine • endoderm: digestive organs and lungs • mesoderm: muscles and bones - “Organizer”: blastoporal lip; tissue that induces differentiation of another tissue - Spemann took blastoporal lip out of donor and into host so that the host had two; the resulting organism had two heads and nervous systems: concluded that blastoporal lip is the organizer of the nervous system; dorsal lip cells could organize the host cells to form a new body axis - Spina biﬁda: failure of neural tube to close caudally; nervous tissue is exposed; treatable, may result in motor deﬁcits - Anencephaly: failure of neural tube to close rostrally; little brain tissue above midbrain, forebrain is mostly absent; brain tissue is exposed; almost always fatal as fetuses or shortly after birth - Hydrocephaly: accumulation of CSF in the brain due to constriction of cerebral aqueduct; aqueduct is narrowing between third and fourth ventricle that allows CSF to ﬂow outside of the ventricles and around the brain and spinal cord; results in increased pressure in the brain and often enlargement of the skull; varying outcomes; gray matter is mostly what is effected; subcortical structures are relatively normal; virtually no neck; shunt can relieve pressure but is risky; can affect only one side of the brain if the blockage is between the lateral and third ventricles - Folic acid (folate) is essential for normal neural tube development; paternal and maternal folate is involved; folate supplement have reduced neural tube defects from .2% to .06% - Neurons are created once neural tube closes - The basal plate gives rise to ventral motor neurons; the alar plate gives rise to dorsal sensory neurons; separated by Sulcus limitans - Lateral horns give rise to sympathetic neurons - The roof plate and ﬂoor plate give animals bilateral symmetry and are important for neurons that cross the midline - Roof plate has corpus callosum ﬁbers - Mitosis of neurons takes place in the ventricular zone - Horizontal cleavage signals the end of division for a particular cell; signaling proteins split so each cell has only one signaling protein - Signalling proteins: numb and notch-1 - Rules for the birth of cells in the CNS: • Proliferation begins in cervical segments and proceeds rostrally and caudally from there; follows behind closing of the neural tube • Large neurons are born ﬁrst. First- born neurons differentiate into Golgi Type I neurons; set scaffolding of nervous system - Golgi type I: large excitatory projection neurons; Golgi type II: small inhibitory interneurons • Motoneurons are born before sensory neurons; sensory neurons follow motoneurons • Interneurons are the last neurons born because of their smaller cell bodies (largely Golgi Type II) • Glia proliferate after neurons, with the exception of radial glia; ﬁrst glia cells are born while neurons are ﬁnishing genesis; radial glia cells serve as guides for hippocampus migration of neurons - Ventricular layer= ependymal layer= proliferative zone= neuroepithelium= birthplace of CNS cells - Mantle layer= newly born, post-proliferative cells - Alar plate: second-order sensory neurons, interneurons; cells are organized in layers - Basal plate: motoneurons, pre-motor interneurons; cells are nuclear - Sulcus limitans: lateral proliferation- free zones; delimiting alar and basal plates - Roof plate: dorsal midline; proliferation- free zone - Floor plate: ventral midline; proliferation- free zone - Neocortex and hippocampus neurons are organized as sheets: derived from alar plate - Cerebellum is derived from alar plate; Rhombic lip gives rise to cerebellum cortex - 4th ventricle is open until 6th week of gustation - 3-4 weeks of gustation: 3 primary vesicles (forebrain, midbrain, hindbrain) - 5-6 weeks of gustation: 5 secondary vesicles (telencephalon, diencephalon, mesencephalon, metencephalon, myelencephalon) - 6 weeks: brainstem becomes more aligned with spinal cord as pontine ﬂexure bends • cerebellum develops on pontine ﬂexure; closes off 4th ventricle • cephalic ﬂexure is maintained: results in brain being at a 90 degree angle from brainstem; neotenous feature - neoteny: maintaining juvenile features in adult animals - Regressive events in the development of the nervous system: • Synapse withdrawal: neurons make more synapses than needed in development, unneeded synapses are withdrawn Axon retraction: neurons make projections to distant parts of nervous system in • development, then axons are retracted • Programmed cell death (apoptosis): death of neurons or population of neurons Development of the CNS: Cellular Mechanisms - Axons must ﬁnd their way to their targets, recognize and respond to midline of the body, recognize like-axons (form bundles), leave pathways, form topographical map, form synapses, match to target sizes - Sciatic nerve is a bundle of nerves in the lower back; as the bundle continues down the legs axons split off from the main trunk - Two classes of chemical factors: may be substrate-bound (membrane bound) or diffusible substances (free in extracellular ﬂuid; form chemical gradients); chemicals can act as both neurotrophins and neurotropins • Neurotropins: guidance cues; lead axon to target • Neurotrophins: nourishing factors; growth and well-being of neuron - Physical, electrical, and temporal variables also play roles in axon guidance and formation of connection • Physical cue: nerves follow blood vessels • Temporal cue: order in which structures grow - Chemical cues are more directly gene products than other variables (easier to study) - Neuroblasts are apolar neurons - Neuron ﬁrst forms many minor processes, one grows to become axon; no differentiation about destined axon to other processes - If an axon is cut, another minor process (dendrite) will grow to become an axon - Contact guidance: mechanical and substrate bound neurotropins; lend a directional component to the axon - Diffusible substances: AKA chemotaxis or chemotropism; attraction to certain chemicals - Guideposts: lead neurons - Timing: order in which neurons are born and axons grow - Galvanotaxis: electrical guidance - Axons in early development do not have terminals; they have growth cones: critical for extension of axons; growth cone leads axon to its destination - Growth cone have ﬁlopodia (extensions of growth cone) and lamellopodia (valleys in growth cone); form “ﬁngers” that stick to substrates to pull axon forward to grow - Paul Weiss (1920s) described growing axons; demonstrated axons cannot be pushed forward by cell body, must be pulled forward (by growth cone); he grew neurons in petri dishes and the axons grew along scratches in dish - Filopodia feel for something adhesive (a substrate) to grab to and pull growth cone forward; some substrates are more adhesive for certain neurons - Growth cone may split into two directions (common at the midline) and one will eventually retract to join the other; growth cone selects which direction to go in by gradients, substances secreted, stickiness of substrates, etc - Guidepost: diffusible or bound; highly localized, near target; no chemical gradient - Contact guidance: substrate adhesion - Chemoafﬁnity (attraction to chemical) and chemorepulsion (repulsion from chemical) - Sticky surfaces preferred are by axons - Growth cone collapses when it reaches its target (ﬁrst step of neurogenesis) - Rita Levi-Montalcini was the ﬁrst to hypothesize the existence of neurotropic and neurotrophic factors (1949); dubbed the neurotrophic factor she discovered “nerve growth factor” (NGF) - NGF acts on neural crest cells and central cholinergic cells. For 35 years, the only known neurotrophin/ neurotropin. NGF is now known to be only one of many diffusible tropic and trophic factors. Growth cones may respond either with positive chemotaxis (attraction) or negative chemotaxis (repulsion) - “hemmed in”: repulsion on two sides forcing the neuron to stay between them - NCAMs: attract other neurons, “Nerve Cell Adhesion Molecules”, homophilic (attracted to similar neurons) - Polysialic acid: contact repellent neuron; glycoprotein interferes with adhesion of NCAMs; allows neuron to continuing growing - Netrins: diffusible and membrane bound; expressed at midline; can attract or repel on the same axon depending on receptor expressed by growth cone (Attract at DCC receptor; repellent at UNC receptor); from Sanskrit “to guide” - Semaphorins and slits: studied at midline; from Greek “sign-bearer” - Guidepost Theory: there is a series of discrete intermediate targets between neuron cell body and its target tissue; must encounter each target to stay on track; ﬁlopodium comes in contact with guidepost cell; guidepost cell also alters expression of genes in the neuron so that different receptors are expressed in preparation for the next guidepost cell - Pioneer neurons: ﬁrst to traverse a pathway; their axons have a unique ability to “read” guidance cues; other neurons are follower neurons, do not have the ability to read guide cues; if pioneer neuron is destroyed, the others will not ﬁnd their way to the target cell - Control of decussations at the midline: chemorepulsion: roof plate releases repulsion cues; ﬂoor plate releases repulsion cues; axons are attracted to the midline - Singular cortex neurons are pioneer neurons for the corpus callosum - Glial wedges and sling cells “hem in” to prevent neurons from crossing the midline - Acallosal: do not have corpus callosum; cells do not cross over midline of the body; acallosal mice were given a glial sling and neurons were able to cross the midline - Slightly fewer than half of retinal neurons cross over midline in humans - Changes in gene expression for netrin and slit receptors mediate behavior of the growth cone at the midline; after a neuron crosses the midline, repulsive receptors are expressed so that it continues to move away from the midline - DCC: attractive receptor for netrin1; “Deleted in Colorectal Cancer”; a tumor- suppressing gene and its product - UNC5: repulsive receptor for netrin1 - Robo: silences DCC attractive receptors while axons are crossing midline; “roundabout” - Topographical mapping: • Marcus Jacobson: neuroembryologist; tried to identify fate maps in developing nervous systems; describe topographical mapping and did early experiments; did work on frogs • Roger Sperry: psychologist; did work on mammals - If you rotate a frogs eye 180 degrees (severing optic nerve), the optic nerve will regenerate; with the regenerated eye, the frogs prey catching behavior is inverted; vision is rotated 180 degrees; same results with any degree of rotation - Chemoafﬁnity hypothesis: there is a matching of chemical signals between eye and optic tectum of frog (analogous to superior colliculus in mammals); thought that axons regrew in new rotated eye back to original myelin sheath so that eye sight would still the same, but the axons grew directly to tectum, inverting their eye sight • found that chemoafﬁnity works as repulsion; lower number of receptors, more ligands; repelled from the wrong target instead of being attracted to correct target - Frogs gain optic neurons in a circular fashion but tectal neurons are in a crescent shape, but can maintain topographical map • desyanpses: synapses are broken and form new synapses • synapses shift as more cells grow to maintain topographical map • human topographical map for hearing shifts as we develop and our head shape changes - In somatosensory cortex, there is a topographical map of our body; map changes throughout life; can change map within hours through training - Silent synapses: not normally active but present; can be activated if they are needed - RGC Speciﬁcation: Jacobson rotated the eyes of tadpoles before retinal ganglion were established • behavioral responses were reversed if done after stage 31 in development • behavior was normal if done before stage 28 • if done at stage 30, dorsal-ventral responses were normal but anterior posterior behaviors were revered • concluded that anterior-posterior were established between sage 28 and 30; each axis is developed independently and at slightly different time - If an eye in one stage was placed in an embryo of a different frog, it was the stage of development that the eye was in that determined the outcome (not the stage that the new frog was in); eye determines outcome even if placed somewhere else in the frog (did experiments by putting eyes in the frog’s tail) - Galvanotaxis/ Neurobiotaxis: electrical guidance; currents in the embryo can be directly measured to determine rostral-caudal direction - Neurotrophins: nourish neurons • apoptosis: “programmed” cell death • axon retraction: elimination of transient collaterals • synapse elimination: reduction in synaptic arborization - Apoptosis: cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, involution of the cell • one regulator of apoptosis is concentration of Ca2+ in cell - Necrosis: cell swelling, chromatin digestion, disruption of cell membrane and organelle membranes, DNA hydrolysis, vacuolation of ER, organelle breakdown, lysis, inﬂammation due to release of cell contents - NGF in TrkA is lost in Alzheimer’s; “nerve growth factor” - TrkC: parasympathetic nervous system - Neurotrophins are synthesized then processed by protease cleavage to generate the mature, processed ligands. All the unprocessed neurotrophins bind with high afﬁnity to the P75 receptor - P75 is death receptor; stimulation initiates apoptosis - One theory: Autophosphorylating: NGF phosphorylates TrkA receptors and a domino effect continues stimulation from growth cone to the cell body - Second theory: Retrograde effector: local signals emanating from Trk receptors in distal axons are themselves propagated to the cell bodies where they promote activation of cell body-associated Trk and consequently cell survival - Effect of target size manipulation on neuronal population: can be regulated by trophic factors; more neurons than are needed are present initially; larger target sizes have larger neuronal populations - Axon retraction: selective loss of an axon collateral; requires that the neuron has multiple targets, each of which provides essential trophic factors; loss of any single target is insufﬁcient to cause cell death; the remaining trophic factors from remaining targets is adequate to assure cell survival - Ipsilateral track in spinal cord is withdrawn; contralateral track maintains - Axon retraction is distinguished from withdrawal of processes of axons of growing neurons; axon retraction is restricted to withdrawal after functional synapses have been made; details poorly understood, but as with cell death, is thought to be mediated by neurotrophic interactions between the neuron and its target; electrical mechanisms may also contribute - Transient collaterals may serve functions other than information processing: ipsilateral corticospinal tract • • visuo-spinal projections - Synapse elimination: the rule when a neuron has multiple targets and is in competition with other neurons; the principal function of synapse elimination is reﬁnement of pattern of innervation (ﬁre together wire together); apoptosis results when a neuron is totally (or nearly so) deprived of its target; the principal function of apoptosis is the regulation of neuronal number - Neonatal muscle ﬁbers have many neurons connected to them; they are eliminated so that each muscle ﬁber is only connected to one neuron; each post-ganglionic cell receives information from only one pre-ganglionic cell after excess connections are eliminated - Inhibitory synapses: increasing Cl- concentration would lower Cl- potential; when Cl- channels are opened, it would become excitatory instead of inhibitory; therefore GABA is excitatory in new cells (inhibitory in older cells) - Activity-dependent ACh receptor clustering: receptors cluster under receptor that received the most ACh - In synapse elimination: • Trophic factor: secreted by target; de-synapsis following axotomy (loss of axon) • NO: nitric oxide: retrograde messenger; activates presynaptic 2nd messengers • Presynaptic Ca+ acts with trophic factor - Ca2+ / Caherin: adhesion - Ca2+/ Calmodulin: presynaptic differentiation into mature synapse in the presence of trophic factors - Synaptogenesis: “Ready, set, go” hypothesis 1. Guidance to target; trophic factors, diffusible and bound 2. Synaptic priming: once axon reaches target, secretion of priming factors by target cells and surrounding glia; growth cone collapse, adhesion; “ready” 3. Induction: synaptic specializations by other NCAMs (neural cell adhesion molecules); pre- and post- synaptic; “set” 4. Synapses begin rudimentary function; before maturity; “go” 5. Stabilization/ destabilization: maintaining or not maintaining synapse (based on activity) - Speciﬁcation: “labeling” of cell’s identity; for example, position of RCGs in retina; Jacobson’s work - Recognition: ligand/receptor interactions; attractive or repulsive; for example, RGC to optic tectum • Miner’s skin rotations in amphibians: rotated skin on tadpoles; ﬂipped dorsal and ventral skin; dorsal skin was white, ventral was green, opposite of normal frog; if you put acetic acid on a frogs skin, frogs are expected to wipe it off; after skin was rotated, the frogs still wiped on the correct side; once frogs grew more, the response switched to wiping the wrong side of the skin; Miner suggested cells become labeled by periphery; sensory neurons that used to innervate belly now innervate back skin and vice versa; periphery alters central connectivity - Induction: axon induces differentiation of target • barrel ﬁelds: whiskers of a rodent, cat, dog, etc are organized in a way speciﬁc to that species; cortex contains “barrels” for whiskers; if whiskers are removed (cut or immobilized), the respective barrels disappear; barrels depend on stimulation of whiskers; cortical organization is determined by sensory input • taste cells: nerve ending grows and causes differentiation of cells; different axons induce different taste cells; sweet vs. salty; axons were severed between CN 7 and salt receptor, and CN 9 and sweet receptors; experiment crossed these axons so that 7 innervated sweet receptors and CN 9 innervated salty; CN 9 induced salt receptors and CN 7 induced sweet receptors; tissue determines type of receptor, not CN; switching cranial nerve connectivity did not change the type of taste bud that resulted • Herbst and Gandry corpuscles: found in beaks of birds; experimenters took beak skin and transplanted it into ﬂank (back) of chick; beak skin was innervated by the spine, and corpuscles formed instead of peripheral cells; location of the tissue did not change the type of cells that resulted - Sensory layers in brain have large input layers and thin output layers; motor layers have thin input areas and large output layers - Garraghty experimented on ferrets because they are still immature when they are born; experiment eliminated some targets for lateral geniculate nucleus; ablated superior colliculus; lateral geniculate nucleus, superior colliculus, and inferior colliculus became smaller; retinal ganglion went to MGN instead of SC; MGN receives information from retina instead of inferior colliculus MGN reacted to light instead of auditory information in new organization; animals • could see with auditory cortex; visual resolution wasn’t as good as normal ferrets, but was still good; orientation columns formed in the auditory cortex; auditory cortex became organized in a way typical of visual cortex, with two dimensions instead of one, like a normal auditory cortex - Timing: ﬁrst introduced by Paul Weiss as a mechanism to explain orderly innervation of target innervation of muscles by spinal motoneurons as topographical in limb-innervating • segments; if muscles are moved, the nerves will still innervate the ﬁrst muscle available in orderly fashion • rostral to caudal motoneurons innervate proximal to distal muscles; mapping is maintained even after limb segment substitutions, additions, reversals, or transpositions; chicks with legs and wings switched ﬂapped legs (like wings) and moved wings like legs; spinal cords were reversed in chicks and got the same result (movements were reversed) • supernumerary limbs: extra limbs moved near the original limb; show “homotypic” (similar) responses if innervated by lumbar segments; if placed too far from original limb sight, do not “reprogram” thoracic segments Epigenetics - Genetics: the study of heritability; a term coined in 1891 by biologist William Bateson; was never formally deﬁned as a hypothetical mechanism of inheritance; as we now know, a gene is difﬁcult to deﬁne - Mendel call genes “inheritance factors” - Current usage of “gene” is in reference to a portion of a DNA strand that codes for a particular protein - “epi”: from Greek meaning “on” or “above” - “genesis”: from Greek, meaning “origin,” “creation,” or “generation” - “Epigenesis” means “above genetics,” or inﬂuences in addition to the genome; more simply, it refers to genes and environment interactions; now implies mediation via gene expression - Epigenesis includes environmentally-induced changes in gene expression but also includes possible interactions between the environment and the genome that do not alter gene expression; deﬁned by phenotype, not genes or environment - Early concepts of epigenesis implied that the fertilized egg contains building materials only, somehow assembled by an unknown directing force; this contrasted with the then prevalent notion of preformationist, which was a widely held belief prior to the 1750s - According to preformationists, a gamete (egg or sperm) contained a perfectly formed embryo that simply grew (became larger) - Current ideas of development are epigenetic in concept, but far more is now known about what directs growth and differentiation - To speak of the genome as a blueprint is simply “gene-speak” preformationism - Genes code for proteins; expression of genes (which are produced and when) is largely a result of environmental events • induction by surrounding tissues (notochord inducing neural tube) • regulation of genes by internal environment (axon guidance) - There is no “hard-wired” master control panel directing development; sequence of local patterns in which one step in development is a subunit of another; each step in the developmental hierarchy is a necessary preliminary for the next - Development is four-dimensional; very early in development, most environmental events controlling gene expression are internal; later, external environments exert control over gene expression as well - Second deﬁnition used in genetics: much more restricted meaning than the ﬁrst usage; alterations to the DNA, other than changes to the genes themselves (i.e. nucleotide sequence), that • are passed on with cell division • can change normal gene expression • can be caused by (early) experience - Epigenesis in this second, narrower, mechanistic sense has long been recognized as essential for tissue differentiation and organogenesis - The external environment can also activate or silence genes, leading to different phenotypes, and that these modiﬁcations can be transmitted across generations, i.e. inheritance of acquired characteristics; this is not Lamarckian in that the genome itself is unchanged - Transcriptional regulation: histone modiﬁcation: chemical modiﬁcation of histone proteins in the nucleosome; the nucleosome is DNA wound around histone proteins - Transcriptional regulation DNA Methylation: methyl group (CH ) added to DNA at CG dinucleotides • 3 - Reduces/prevents transcription - Tissue speciﬁc - Important in embryogenesis & tissue differentiation - zygote largely unmethylated - series of methylations leads to tissue differentiation - Possible source of trans-generational epigenetic transmission • Histone Modiﬁcation: Inﬂuences “density” of DNA packaging in chromosomes - Inﬂuences transcription - Cocaine & amphetamines (and other drugs) cause histone modiﬁcation • Transcription Factors - Transcription factor (regulatory protein): protein or protein complex that enhances or inhibits transcription - Alter gene expression without altering DNA itself; expression is reversibly dependent on presence or absence of transcription factors - Most well-studied epigenetic mechanism is methylation of cytosine on the DNA - If methylation occurs in an active stretch of DNA, especially a promoter region, the associated stretch of DNA (gene) will likely be silenced - Methylation of DNA, and thus gene expression, continues after birth and be inﬂuenced by the broader environment (nutrition, stress, drug use, lifestyle, etc.) - Heritability is not a calculation of, nor is it even an estimate of, the degree to which a phenotypic character is inherited - Heritability is also not a measure of the degree to which a particular character trait is genetic or environmental - Heritability is an estimate of the total population variation of a phenotypic character that is attributable to genetic variation. H = V /g t - Variance of the population (V t) is partitioned into variance due to genetic variability (V g), that due to environmental variability (V e) and their interaction (V gxe V = V g + V e + V gxe • - It is not possible to measure Vgxe directly, and is assumed to be negligible. Thus, Vt= Vg + Ve - It is difﬁcult or impossible to eliminate Ve, whereas Vg is easy to eliminate through the use of inbred strains, clones, or identical twins. Thus, Ve is estimated as the residual variance in genetically homogenous populations,, in which Vg=0 So, Vt= 0 + Ve • - Since we can easily measure Vt directly and have an estimate of Ve from our genetically homogenous population, it is a simple matter to calculate Vg in the random population by subtraction: Vg= Vt- Ve - Assumptions for this formulation of heritability: • genetic and environment affects on phenotypic variability are additive • that gene x environment interactions are negligible • that Ve is the same for inbred and outbred strains - Heritability quotients depend as much on environment variation as they do on presumed genetic variation - Heritability estimates are limited in that they apply only to the population represented in the sample and cannot be generalized to other populations - Twin studies: independent variables • Genetic variables: monozygotic vs dizygotic vs siblings vs unrelated • Environmental variables: reared together vs reared apart - Twin studies: dependent variables • Concordance: all-or-none; if one member of the pair has the trait, what is the probability that the other shares the trait Correlation: graded trait; is there a difference in the correlation of the trait in pairs in the • different groups - Monochorionic twins: share a placenta - Dichorionic twins: have different placentas - Monozygotic twins who share a placenta have a 60% concordance rate for Schizophrenia; monozygotic tons with different placentas have only an 11% concordance rate - Epigenetic transmission: • Whole Chromosome Regulation (X chromosome inactivation or Lyonization) • Regulation during Protein Synthesis - Transcriptional Regulation • methylation • histone modiﬁcation • transcription factors - Editing regulation • alternative RNA splicing: Different exons are spliced together to give different polypeptide blueprints; can produce variation between species; possibly why number of human genes is so small • exons are nucleotide sequences that are present in RNA products; introns are nucleotide sequences that are edited out during RNA splicing - Pre-translational regulation • “interfering” RNA: A short sequence of single-stranded interfering RNA (“iRNA”) and a complex of proteins and enzymes (“silencing stuff”) binds with mRNA and cleaves it; acts as a “dimmer switch,” reducing translation • Regulation after protein synthesis; many mechanisms - Protein activation/deactivation • Phosphorylation: add a phosphate group • Acetylation: add an acetyl group • Alkylation: add an ethyl, methyl group • Ubiquitination: adding the protein ubiquitin to an existing protein instructs cellular machinery to degrade/ destroy the protein - Epigenetic transmission: two types • Blue print transmission: sequence transmission; transmission of information via the nucleotide sequence (A,C,G,T) • Regulatory transmission: epigenetic transmission; transmission of information via gene regulation; transmission of genetics above the sequence of nucleotides (gene methylation and histone modiﬁcation) - Genomic imprinting: The expression (active vs inactive) of a gene depends on which parent transmits the gene; some turned off when inherited from the father and turned on when inherited from the mother; others turned on when inherited from father and turned off when inherited from mother • Mechanisms: methylation, phosphorylation of histones (examples of epigenetic transmission) - Epigenetic transmission: behavioral example: rats either handled or not handled in infancy; early handling reduces anxiety throughout life; offspring of handled rats less anxious; offspring of offspring (grandchildren) less anxious • anxiety is operationally deﬁned as reduced activity in a open ﬁeld Behavioral Embryology - Functions of embryonic and fetal behavior: necessary for normal anatomical and physiological development; serves adaptive functions as a behavior; serves as practice for future behavior; is an epiphenomenon of no particular importance - Coghill’s view of behavioral development: earliest movements are spontaneous rather than elicited; from the beginning, movements are “ mass actions”: all parts of the animal that are able to move, do move; these mass actions become individuated into more localized and discrete movements • Behavior is sculpted from undifferentiated precursor, much like a sculptor chips away all of the stone that is unwanted, leaving the ﬁnal differentiated form - The behavior pattern develops as a regular, orderly sequence of movements, which is consistent with the order of development of the nervous system and its parts - In a relatively precise manner, physiological processes follow the order of their embryological development in the functions of aquatic and terrestrial locomotion and feeding - Behavior develops from the beginning through the progressive expansion of a perfectly integrated total pattern and the individualism within it of partial patterns which acquire various degrees of discreteness - Windle’s view of behavioral development: ﬁrst movements are forelimb proprioceptive reﬂexes, other reﬂexes follow (oral reﬂex appear next); complex behaviors emerge as local responses become integrated with each other • Behavioral development is achieved by putting together small pieces, much the way a machine is assembled - Why do Coghill and kindle disagree species differences, experimental artifacts, and types of behaviors studied - Classes of avian embryonic behavior: • Type 1 behavior: generalized behavior of the embryo; found from the onset of motility until pre-hatching; includes jerky, conditioned movements of the limbs • Type 2 behavior: startle-like behavior; found from the onset of motility thought incubation • Type 3: pre-hatching, hatching; highly organized, not predictable from types 1 and 2; occurs only in the days immediately prior to hatching - Neural plate differentiates in chicks at 28 hours, rats at 7 days, humans at 19 days - Long period of motility before close of reﬂex arcs - First movement is lateral head movement (sideways bending), then “startle,” then generalized movements - First movements begin at about 7 weeks gestation age in humans (fetus is about 1 cm long); mothers can detect it at 16 weeks - Cryptic behavior: exists in development but not after birth - “Fetal breathing”: inhaling and exhaling amniotic ﬂuid - Fetal hiccuping is precursor for breathing - What role does experience play Facilitation: experience causes accelerated development boost in terminal level • achievement, and a combination of accelerated development and an elevated terminal level of achievement • Maintenance: experience must be maintained to alter sensation • Induction: sensation fails to develop if there is no experience - “Visual cliff” : cliff marked with checker board with clear glass at the end, so it appears to drop off but does not - Dark-reared animals showed delayed maturation Transition from the Womb - Birth as a “non-event”: neurobehavioral systems required by the newborn are functional prior to birth (hiccup-like, breathing-like) • “Forward reference” or “environment expectant”: by Weiss; develops anticipating needs, not in response to them; contrasts with “environment- dependent” - sucking, swallowing, “breathing”, “hiccups”, yawning” are forward reference; most occur mid-term - reﬁnement of ocular columns is example of environment- dependent - Other than constraints imposed by the intra-uterine environment (fetuses cannot cry without air, for example), there is little behavioral difference between a late-term fetus and a newborn; newborns are not doing anything new after birth - What distinguishes a newborn from a fetus is that there behaviors are necessary for survival; breathing is functional but necessary in newborn - Initiation of breathing: noxious stimuli; spanking a newborn is not necessary to initiate breathing, and birthing in warm water makes little sense from either a physiological or evolutionary perspective • temperature change is a noxious stimulus • anoxia produced by clamping the umbilical cord • ﬂuid is expelled from lungs during delivery, the remainder quickly absorbed • changes in other sensory inputs - Infants learn the sound of a mother’s voice in utero; infants prefer sound of mother’s voice; do not have preference for father’s voice compared to other males; prefers how mother’s voice sounds in utero to how it sounds outside the womb (proof that newborns learn the sound of mother’s voice in utero) measured preference based on how the rate of suckling changes in response to hearing • different voices - Sucking and swallowing are essential to the newborn, but have been expressed by the fetus - About the only thing not apparent in the fetus that is expressed by the newborn is crying - From a neurobehavioral perspective, birth can be considered to be a “non-event,” unlike, for example, amphibian metamorphosis • better analogy for metamorphosis is weaning - Oxytocin stimulates uterine contractions, but what stimulates oxytocin release • labor is induced medically with an injection of oxytocin - Surfactant protein A (SF-A) initiates birth: makes liquids “wetter”, reduces surface tension; essential for respiration after birth production in lungs of fetus is signal that lungs are developed; SF-A is released into the • amniotic ﬂuid and triggers oxytocin release • begins being produced in mice at 17days, birth at 19 days • begins being produced by human fetus at 32 weeks, birth at 40 weeks - Other than the capacity for independent respiration, there is little relationship between maturity and birth/ hatching - Newborn opossum continues to develop in the mother’s pouch after birth - Precocial: born in a relatively mature state (guinea pig, kittens) - Altricial: born in a relatively immature state (opossum, rats) - Humans are born with relatively mature sensory systems (precocial in that respect); motor systems are very immature at birth, can’t roll over (altricial in that respect) - Changes in total brain weight as a function of age in different animals: brain growth spurts reach a peak much after birth in altricial animals, and before birth in precocial; occurs around birth in humans - Study of survival of animals in pure nitrogen environment (no oxygen) at different ages: death of the animal is determined by survival of the brain; less mature brains can survive longer in oxygen free environments (younger animals of the same species; more altricial animals) • newborn rats survived for an hour in the oxygen free environment with no long-term brain damage • human infants can survive longer in hypoxic environment than older children (fall in pool, etc) - Most caudal parts are more mature, more rostral parts are less mature; more mature requires more oxygen consumption; throughout development, rostral begins to consume more oxygen than caudal - Maturation of myelination moves caudally to rostrally - Neonatal period in rat is ﬁrst 2 weeks; weaning begins at 2 weeks - Newborn rat cannot ﬂip himself over (“right himself”) when placed on their back in a supine position - Rats 0-4 days old can right themselves even if given a caudal transection - After 4 days old, more caudal transections in rats make it increasingly harder for them to right themselves - At four days, the caudal parts of the brain begin developing connections with rostral parts of brain; before four days, the caudal areas can act independently; after four days, the caudal parts of the brain cannot operate without connections to the rostral parts - Caudal brain organizers itself as rat ages, and the further they have organized, the more harmful transection is - Brains transected at four days could develop normally, with little change based on where it was cut; after four days, the more caudally the cuts were made, the more hinderance to development there was - In an experiment where spines were cut, there was no hinderance to development if the surgery was done between 0 and 12 days; at 15 days, there was loss of control The Neonatal Niche - Neonates face problems not face by adults; unique to neonates because of their size, anatomy, and physiology - Neonates adopt their own strategies to cope with these unique problems - Coping strategies are both behavioral and physiological - Homeothermy: the physiological and behavioral maintenance of a relatively constant internal body temperature (homeotherms typically show ﬂuctuations, e.g. circadian rhythms, in temperature); humans - Endothermy: physiological and behavioral thermoregulation, but body temperatures may ﬂuctuate widely; - Ectothermy: use only behavioral means to regulate temperature; butterﬂies - Poikilothermy: the ﬂuctuation of internal body temperature closely related to environmental temperature; most ﬁsh - Cold blooded: poikilothermy > ectothermy > endothermy > homeothermy: warm blooded - Heat gain: volume (size) of thermogenic tissue; basal metabolic rate (how active is tissue); shivering or other thermogenesis - Heat loss: surface area; insulation (fat, feathers, hair); panting, sweating, etc - Infants have small bodies for heat generation and heat loss - Peripheral thermoreceptors are found in the skin; related to behavior - Central thermoreceptors are found in the anterior hypothalamus; controls seasonal ﬂuctuations in body temperature - These thermoreceptors are important for behavioral and physiological thermoregulation both in the short term and in the long term - Short term thermoregulation is regulated predominantly by autonomic and somatic motor activity - Autonomic: • shivering when cold, sweating when hot peripheral vasodilation when hot, constriction when cold • • piloerection (fur standing up) and panting in non-human mammals • adrenal gland: release catecholamines to increase basal metabolic rate - Somatic: • seeking warm (sun) or cool (shade or water) areas • minimizing or maximizing surface area - Long term (e.g. seasonal or as adaptation to different climates) thermoregulation is predominantly regulated by hormonal regulation of metabolism - Thyrotropin releasing hormone (TRH) is secreted from the hypothalamus to stimulate release of thyroid-stimulating hormone (TSH, also thyrotropin), which stimulates release of thyroxine from the thyroid gland • tissues respond with an increase in their basal metabolic rate - Autonomic and hormonal responses to thermal challenges are mediated primarily by the medial pre optic area - Behavioral responses to thermal challenges are mediated principally by the lateral hypothalamus - Maintenance of thermal homeostasis is arguably the greatest factor inﬂuencing both energy balance (via calories spent for thermogenesis and the need for fat stores) and water and mineral balance (water lost via evaporation through the skin, metabolic processes necessary for homeothermy and panting) - Allometric growth: parts of the body grow at different rates - Problems faced by altricial neonates: • sensory and motor immaturity CNA immaturity • • Physiological immaturity (autonomic control, homeostatic regulation) • morphological immaturity (small size, allometric growth) - Thermoregulatory problems of altricial mammals: • cannot shiver • has precarious energy balance; do not have extra energy because all energy is used on growth • small size • cannot vasoconstrict in response to cold • no hair; humans depend on adults for clothing • little body fat and thin skin for insulation - The problem with being small: surface area to volume ratio; if length increases linearly, surface area and volume increase exponentially; surface area increases more slowly than volume surface area to volume ratio increases with age; more surface area per volume • • at ambient room temperature, a newborn rat would have to increase its metabolic rate 6-fold to maintain its core temperature • newborn rat and dead rat lose heat at the same rate when put in cold water • hypothermia is used as anesthesia for rats; hard to overdose and less bleeding - Brown adipose tissue (BAT) is not used as long term storage for nutrients but is metabolically active; densely vascularized; dense mitochondria; increased metabolism allows newborns to raise body heat - Bears use brown fat during hibernation - Brown fat accounts for 5 to 6 percent of the body weight of the newborn rabbit. It is concentrated around the neck and between the should blades - Human infant at birth has a thin sheet of brown adipose tissue between the should blades and around the neck, and small deposits behind the breastbone and along the spine - Brown fat is located near heart and spinal cord to maintain these most vital organs - Basal metabolic rate (BMR): minimum metabolic rate at thermoneutral temperature; mid 70s to mid 80s in naked adult humans - Cold stress will result in increased metabolism which will lead to increased oxygen consumption, hypoglycemia, hypoxia, and respiratory distress - Newborn rats can be cooled until their heart stops and then rewarmed to start it again; adult rats cannot start their heart back up; newborn humans can survive in water longer than adult humans - In mild cold temperatures, newborns will attempt to thermoregulate; in very cold temperatures, they will shut down their metabolism - Rats who are transected at level 3 are unable to maintain their core temperature in warm environments; have thermogenic response and heat up - Transected animals have an increased oxygen consumption in warm temperatures; intact animals show thermogenic response but then shut down to return to BMR - Blood glucose levels are initially higher in transected animals; blood glucose levels decrease in transected and intact animals, but are overall lower in intact animals - Intact animals shut down as blood glucose level drops because they are able to monitor glucose stores; transected animals maintain high metabolism in both temperatures even as glucose stores decrease - Intact animals injected with insulin do not illicit a thermogenic response; made them hypoglycemic - Lesions in hypothalamus do not affect animals; mechanisms responsible for thermoregulation is located somewhere rostral to transection but not in hypothalamus - Temperatures were taken rectally; rectal temperature is most accurate in adult rats, but not in newborns; newborns are warmest under brown fat (upper back) and rectal temperatures are coolest - Oxygen consumption (metabolic rate) compared to environmental temperature of differently aged rats: • 21 days old:steady decrease in metabolic rate as temperature decreases • 23 hours old: increase of oxygen consumption at 30 then declining as temperature decreases; have nursed so they can produce thermogenic response • 4 hours: temperature does not have an affect on metabolic rate; they do not have energy to produce thermogenic response because they have not nursed yet - 5- 10 day old rats require huddling with littermates to stay warm; they have not developed thick fur or developed thermogenesis; older rats are also larger - 5 and 10 day old rats lose temperature rapidly if separated from litter; temperature decreases more slowly if with only three littermates - 15 and 20 day old rats can maintain their temperature when alone and when with three littermates - Newborn rats thermoregulate as one group when they huddle (clump) - Huddles form convection currents when moving around their clump; positive thermotaxis; if a pup is cold, they dive into the top of the huddle, pushing others out; those who were pushed out jump back to top when they get cold - In warmer temperatures, the current reverses and rats jump out of top to get to the cooler area - Rat- shaped robots programmed to move randomly resemble real rat behaviors; formed clumps; both got stuck in corners; shape of rats facilitates clumping behavior - Reptiles move to thermoneutrality: area between hot and cold - Reptiles and infant rats cannot generate a fever when injected with bacteria - Fish and reptiles spend more time in heat when injected with infectious agent - Newborn rabbits prefer higher temperatures (40.5 degrees C) when injected with pyrogen vs. saline (36.5 degrees C) Human Infants: - Heat Production • The ability to increase metabolic rate in response to cold stress begins around 28— 30 weeks postconceptional age. Postconceptionally older infants can increase heat production, but the response is weaker than in the adult. 4’16 • Limited stores of metabolic substrates (glucose, glycogen, fat, etc.) • Heat production needs met primarily through non-shivering thermogenesis; however, the amount of brown fat stores are inversely related to gestational age. • Heat production obligates oxygen consumption, challenging the immature cardiovascular and pulmonary systems. • Large surface-to-mass ratio and large surface heat loss relative to heat-producing ability result in high metabolic rate. Large evaporative loss due to status of skin maturation; evaporative loss itself may • exceed heat production abilities. • Shivering response not well developed. Cannot initiate increased tone and shivering to increase heat production. - Insulation • Limited layer of subcutaneous fat, limited development of muscle and other tissues that provide insulation. • Small body diameter results in thinner layer of still air boundary layer, reducing insulation through this mechanism. - Vasomotor Response • Competent abilities to regulate skin blood ﬂow documented in infants weighing >1 kg; however vasoconstriction abilities are outmatched by propensity for heat loss.4 - Sudomotor Response • Sweat production observed in infants of 29 weeks gestational age; maturation of response enhanced by extrauterine development. Response is slower and less efﬁcient than in older child or adult, and occurs at a higher environmental temperature.24 - Motor Tone and Activity Lower postconceptional aged and ill infants prone to decreased motor tone and • less activity, resulting in decreased heat production. Infant with poor tone cannot use ﬂexion posture effectively to reduce surface area and hence heat loss. - Behavioral • Limited ability to effectively communicate thermal needs or thermal comfort to caregiver. • Cues are subtle and nonspeciﬁc. • Cannot use volitional actions such as altering clothing, increasing ambient temperature, using motor activity to increase heat production, drinking warm or cool beverages to modify temperature. - Brown fat is stimulated by sympathetic innervation (not blood-borne signals) - Ultrasonic Vocalizations in newborn rats: emitted by isolated pups in ﬁrst two weeks • isolation necessary and sufﬁcient condition to elicit vocalization • • retrieved by dam; mom rat gets newborn if they are making vocalizations • called “isolation distress”: distress implies that it is an emotional response; this would make a good model for separation anxiety in humans - Vocalizations as “cries” • implies emotional response analogue of separation anxiety • - Alternative explanation • part of thermogenic response; physiological reason not psychological • exaptation: adaptations that are selected as a consequence of another; ultrasonic vocalization may be used as part of thermogenic response, but also results in dam (mother) retrieval • not “cries,” but more like “grunts” • laryngeal braking: closing larynx; prevent passage of air - enhances gas exchange - facilitates return of blood to heart - Valsalva maneuver: tightening of abdominal muscles; like humans grunting when lifting weights or playing tennis; abdominal compression response - Thermoregulation summary: Problem: Cannot shiver to produce heat Solution: Brown adipose tissue (BAT) Problem: Precarious energy balance Solution: Thermoregulate over narrow temperature range, shut down to conserve energy if temperature is out of range, tolerate lowered temperature Problem: Small size, large surface area:volume ratio Solution: Clump to reduce surface area:volume ratio Problem: Poor insulation Solution: Clump, nest insulation (mother builds nest), thermotaxis, ultrasound response to isolation, mother is source of heat while nursing, most rodents live in burrows for warmth (also to escape predators) - Thermogenesis is not a precursor to adulthood; it is speciﬁcally neonatal; it persists in adults as a machismo of hibernation - Huddling may be both for thermogenesis and as a social behavior Suckling - Source for nutrition as well as ﬂuid - Olfactory cues are important for pups ﬁnding nipple; washed mother has much less attaching by pups than the unwashed mother; there is an odor on nipples that help them recognize it • if washed extract solution was put back on the mother, they would attach; they would also attach if pup saliva was put on nipples; also would attach if amniotic ﬂuid was put on nipples (washed extract: the water used to wash mother’s nipples) - pups inhale and swallow amniotic ﬂuid in the womb; after birth, mom licks pups and then licks nipples so that the nipples have amniotic ﬂuid • virgin female saliva and mother’s urine on nipples did not elicit attachment • “nipple extract”: swabbed from another mother’s nipples elicited attachment; if other mother was on a different diet, there was not attachment • pups attach if washed mother is injected with oxytocin • ligated nipples: tied so milk is not released; if mother with ligated nipples is injected with oxytocin, pups still attach • if injected with atropine, there is less attachment - Dimethyl disulﬁde is common to the effective solutions, and when a solution of dimethyl disulﬁde dissolved in distilled water is painted on the nipple of a washed dam, attachment is reinstated - In experiment, pups were exposed to citral (citrus smell) prenatally and/or postnatally and nipples are citral scented • if not exposed at all: no attachment • if exposed postnatally only or prenatally only: no attachment if exposed both pre and postnatally: attachment • - Nipples are not citral scented; dimethyl disulﬁde scented nipple • if not exposed to citral at all: attachment • if exposed to citral only post or prenatally: attachment • if exposed to citral post and prenatally: no attachment to dimethyl disulﬁde scented nipple - Canulas are placed in pups mouths and dam in anesthetized (cannot eject milk); anesthetized dams produce milk if injected with oxytocin - Pups form tight seal around nipple and suck in bursts; change is poster of pups once milk is released (arched back, limbs extended, stretched, tail lifted) to facilitate milk delivery; increases negative pressure (sucking); response disappears between 10 and 15 days - Pups show no preference between a non-nutritive nipple (does not give milk, fake nipple) and a nutritive nipple (real nipple) up to 12 days of age. - Pups that have gone without food for 23 hours show no more latency (time to attach to a nipple) than those who have not been deprived up to 13 days old. After 14 days old, the deprived animals will suckle quickly. - Older pups (over 13 days) have a preference for nutritive over non-nutritive nipples, so if a nipple stops producing milk, the pup will switch to a different nipple. At younger ages, the pup will stay on the nipple even if it is not producing milk. - Suggests suckling is independent of the reward of food; suckling is rewarding itself, it is not only done to get milk - Experiment measured if deprived pups would lick milk from the ﬂoor; in warm temperatures, the pups would lick the milk; in cold temperatures, they would not lick it - The temperature of the pup does not affect their tendency