final - discussion

What are the advantages and current limitations of retinal organoid modeling?

"Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, serving as effective in vitro models of retinal development." - derived from induced pluripotent stem calls; can form retina in a dish - organoids can recapitulate a lot of what we see in normal retinogenesis a. normal cell layers, 7 major cell types Advantages: (1) it mimics the 3D structure of the retina, (2) it correctly uses temporal and spatial cues to produce all of the retinal layers and 7 major cell types, (3) forms all main photoreceptor subtypes, (4) forms adjacent structures such as adherens junctions, microvilli, and Muller glial cells, (5) can develop some synaptic maturity (to transfer information), (6) possible use for transplants to treat retinal diseases, (7) can be used to mimic retinal disease in order to understand disease mechanism or to test therapeutic strategies. Limitations: (1) incorrectly produces positional cues so photoreceptors are seen apically and basally (not just apically), (2) cannot form separate specialized fovea, (3) quality of adjacent structures varies, (4) represents an immature/incomplete development stage, (5) reproducibility and staging, (6) limited integration and functionality of transplants.

In the attached figure, a 5-ms depolarizing current pulse was injected into the soma, which produced a single action potential that was recorded in the cell body. Right after that, the distant dendrites of the neuron were activated, which generated a dendritic spike, which propagated to the cell body and resulted in two additional somatic action potentials. What would happen to (a) the first, and (b) the second two action potentials if you blocked the dendritic action potentials?

(a) If you blocked the dendritic action potentials, then the first action potential would be unaffected and would still occur because the injected current in the soma would still produce the first action potential regardless of the following dendritic action potentials (b) However, the action potential produced from the injection into the soma allowed back-propagation of the action potential combined with the activation of the distant dendrites to generate the dendritic spike which then caused the two additional action potentials. If the dendritic action potentials were blocked, then you would not get the two additional somatic action potentials because the threshold would not be reached

What are the similarities and differences in the organelles and cellular location of protein synthesis for calcium/calmodulin-dependent protein kinase II (CaMKII) subunits and metabotropic glutamate receptors? Why?

(membrane protein) Glutamate receptors are localized to the plasma membrane of postsynaptic dendrites and interact with the neurotransmitters released from the presynaptic neuron to transmit information. Transmembrane proteins are synthesized in the rough ER before passing through the Golgi apparatus and fusing with the plasma membrane. (cytosolic protein) The mRNAs that encode for CaMKII subunits are targeted for dendritic processes and translation is localized to the dendrites using clusters of free ribosomes (polyribosomes) in the cytosol. This localization makes sense because the proteins produced in this local protein synthesis function in dendrites and postsynaptic sites. - Both known to be synthesized in dendrites, but most in cell body and transported - New evidence for local protein synthesis in axons too!

Which mental trait has the lowest heritability (contribution of genetic differences to trait differences)?

(rMZ - rDZ) x 2 = ? Heritability is the difference between the correlations of monozygotic and dizygotic twins multiplied by 2.

1. Action potential threshold 2. Action potential 3. Subthreshold stimulus 4. Suprathreshold stimulus

1. the membrane potential that must be met or exceeded to generate an action potential 2. a large and transient depolarization of the membrane potential above the threshold 3. a stimulus that does not generate an action potential 4. a stimulus that generates an action potential

For each labeled point (A-D) on the action potential shown in Figure 2, state whether the conductance through voltage-dependent Na+ and K+ channels is low, high, or no conductance. Explain. [Note: For this answer, ignore conductance through leak channels.]

A - low conductance for Na+ or K+ (channels are closed - no depolarization has occurred yet - resting) B - depolarization is due to high conductance of Na+ (Na channels open - increase in Na+ conductance causes Na+ influx), low conductance for K+ yet (lags behind Na+) C - low conductance of Na+ (inactivating of the Na+ channels), high conductance of K+ (K+ open, increase in conductance of K+ causes K+ efflux) D - no conductance for Na+ since channels are all closed and inactivated, low conductance for K+ (hyperpolarization - potential resembles equilibrium potential of K)

What is a non-spiking neuron and how does it transmit information?

A non-spiking neuron will never transmit information via an action potential. It will use a graded potential to transmit information instead.

What is the normal flow of information in a neuron (functional polarity)? Contrast the with amacrine processes.

According to Cajal's functional polarity theory, the normal flow of information through a neuron is in one direction where the dendrite and cell body receive information which travels to the axon where information will be transmitted to the next neuron or effector cell. Some interneurons do not have dendrites and an axon, so they are said to have amacrine processes instead. Lacking the normal flow of information, amacrine processes support bidirectional transmission of information. EX. motor neurons in hydra (?)

What is the difference between active and passive transport and how do they work to move solutes across a membrane?

Active transport allows for the transport of solutes against the electrochemical gradient, thus requiring energy from chemical reactions (ATP hydrolysis), light, coupled transport with other solute gradients, etc. to do so. Passive transport allows for the transport of solutes through a channel/transporter in the direction of the electrochemical gradient without needing to use external energy to do so.

Describe the various developmental processes that comprise the "activity-independent" stage of neural development. Which of these stages is particularly sensitive to folate deficiency?

Activity-independent - occurs independent of neuronal firing (so before establishing synapses) The processes that are activity-independent include the establishment of the primordial nervous system in the embryo via (1) gastrulation (development of the three germ layers), (2) neural induction (mesoderm signaling up to ectoderm to induce neural plate), and (3) neurulation (neural plate receives cues to fold into neural tube). Other processes include the initial generation of neurons from undifferentiated precursor cells (proliferative phase with symmetric cell division in epithelial cells with apical and basal polarity to make more progenitors), neural differentiation (asymmetric cell division to create one precursor and one differentiated - neurons or glia), neural migration (signals the migration of neurons to their target areas), neuritogenesis (extension of axons and dendrites), and lastly synaptogenesis (start of wiring by forming synapses). Neurulation is particularly sensitive to folate deficiency as it can leads to problems regarding closure of the neural tube.

The phototransduction pathway in photoreceptors stimulated by light is diagrammed in the figure below. What happens to cGMP in the light and how does this effect the CNG channel and the polarization of the cell? What happens to cGMP, the CNG channel and polarization of the cell in the dark state? If you injected phosphodiesterase into the cell while it was still in the dark, would the membrane become more depolarized or less depolarized, or would there be no change in the voltage across the membrane? Explain your answer.

After light activates rhodopsin, the alpha subunit with GTP activates phosphodiesterase (PDE). PDE hydrolyzes cGMP to GMP, leading to the closure of CNG channels and therefore hyperpolarization of the photoreceptor. In the dark state, PDE is not activated and does not hydrolyze cGMP to GMP, so cGMP levels increase. cGMP then binds to the cytoplasmic portion of the CNG channel and increases its conductance, leading to an influx of cations and depolarization of the photoreceptor. If you injected PDE into the cell while it was in the dark, there would be no change in the voltage across the membrane. Even though you are adding PDE, PDE would not be able to hydrolyze cGMP to GMP because it is in an inactive state since it was not activated by transducin, and the CNG channels would remain open and the photoreceptor would remain depolarized.

In the last unit we learned how one branch of the sensory stimulation evoked in the knee-jerk reflex results in direct stimulation of extensor muscle fibers. Describe the sequence of molecular events, triggered by the action potential elicited in the recipient motor neuron, that ultimately results in neurotransmitter release. What affect would the application of botulinum toxin at the neuromuscular junction have on this process? Why?

An action potential depolarizes the presynaptic terminal to open voltage-gated calcium channels in the presynaptic membrane, and an influx of calcium into the presynaptic terminal triggers the release of neurotransmitters after the vesicles fuse with the presynaptic membrane. The neurotransmitters then bind to postsynaptic target receptors. Botulinum toxins blocks synaptic vesicle fusion with the presynaptic membrane by cleaving SNARE proteins, thus preventing neurotransmitter release and target cell response (EX. muscle contraction).

What is meant by anterograde and retrograde AXONAL transport? Which motor proteins are involved in these types of axonal transport? What components of the cytoskeleton are necessary for this type of transport?

Anterograde transport refers to transport from the cell body to the axon terminal whereas retrograde transport is the opposite, referring to transport from the axon terminal to the cell body. Dynein and kinesins are motor proteins involved in these types of axonal transport. Dynein performs (fast) retrograde axonal transport from axon terminals back to the cell body while kinesins perform (fast and slow) anterograde axonal transport from the cell body to axon terminals. In vertebrate dendrites, both motor proteins can perform bidirectional transport. -Microtubules are necessary for this type of transport. The motor proteins move along the microtubules over long distances such as to the general periphery or towards the cell body.

What is the advantage of having interneurons in terms of information processing?

As an intermediate between sensory and motor neurons, interneurons allow for further convergence (can receive inputs from multiple neurons) and divergence (cascade effect) of information processing. Due to this, increased response complexity and integration is supported. The article mentions other advantages like excitatory or inhibitory switch activity, pattern detectors and generators, and pacemaker activity that interneurons can display.

Figure 3 shows a whole-cell sodium current (bottom trace) elicited by stepping the membrane potential from -70 mV to 0 mV (top trace). The dashed line is 0 nA. Upon depolarizing the membrane, there is an inward current. This whole-cell current is a reflection of the cumulative activity of many individual ion channels. What is the probable state of an individual sodium channel at each point (A, B, and C)?

At point A, sodium channels are closed as depolarization has not occurred yet. At point B, the sodium channels are open and sodium ions are flowing into the cell causing the inward current. At point C, the sodium channels are slowly inactivating, thus preventing further sodium influx and accounting for the decrease in current.

What mechanisms are used to clear neurotransmitters from the synaptic cleft?

Besides neurotransmitters simply diffusing out of the synaptic cleft, there are more efficient methods to clear neurotransmitters from the synaptic cleft. Esterase enzymes function to degrade neurotransmitters - for example, acetylcholinesterase degrades acetylcholine in the synaptic cleft. Reuptake of neurotransmitters can also occur when the neurotransmitters are taken back up into the presynaptic terminal via plasma membrane transporters, where they can fill vesicles again, or neurotransmitters can be taken up by transporters on glial cell plasma membranes.

How does BMP signaling contribute to neural development?

Bone morphogenetic proteins belong to the transforming growth factor (TGF) family of peptide hormones, and they promote epidermal fate rather than the neural program. The outcome of BMP signaling is modulation of gene expression to elicit the epidermal fate from mesodermal cells. So if ectodermal cells are presented with BMP, they will have an epidermal fate. Inductive signals that are necessary for neural induction and differentiation include BMP inhibitors and FGF signaling. If these inhibitors (or FGF signaling) are absent, then the ectodermal cells would form skin and not neural tissue. On the other hand, if BMP is absent, then the ectodermal cells would form neural tissue and not skin. Thus, this negative regulation of BMP allows the neuroectoderm to form.

What are the embryonic origins of the CNS and PNS?

Both CNS and PNS are derived from the ectoderm. Once the neural plate is formed, the neuroepithelium present here fold to form the neural tube which will generate the entire CNS (brain, spinal cord, retina). The neural crest cells of the neural plate border located on the sides of the neural plate migrate away from the neural tube to become the PNS (neurons, glia) and cells that form neurogenic placodes found in PNS of the head region.

What is meant by cell intrinsic and cell extrinsic factors? Describe how they mediate retinogenesis.

Cell intrinsic factors refer to properties of the cell that contributes to its phenotype while cell extrinsic factors refer to components of the cell's environment that act on the cell to influence its phenotype. Both influence cell fate including structure and function. In retinogenesis, intrinsic and extrinsic factors play a role in determination, competence, specification, and differentiation. They promote multipotent retinal progenitors to develop into competent postmitotic precursors. The competent postmitotic precursors then undergo further specification before final differentiation into the adult cell type occurs.

Describe generally how cortical neurogenesis leads to the formation of a 6-layer cortex in mammals and tell me which cortical layer the cells from the second wave of cortical neurogenesis finally occupy.

Cortical neurons arise from progenitors in the ventricular zone of the dorsal telencephalon. Via asymmetric cell divisions, first born neurons form the subplate. The second wave of neurons born pass the subplate and establish the cortical plate and will mature to form layer 6 neurons. After this is established, newly born neurons migrate radially to the appropriate layer. Early born neurons which exit the cell cycle first, remain in the deepest cortical layer 6. The next postmitotic cells will radially migrate past the early born neurons in layer 6 and reside in layer 5. This pattern continues until the latest born neurons migrate to layer 1, the most superficial layer.

How do Na+ and K+ ion channel structures allow them to detect changes in voltage across the membrane?

Each of the four repeating modules in the channel contains six transmembrane segments. The fourth transmembrane segment (S4) consists of many positively charged amino acids. When the voltage across the membrane becomes more positive inside, this repels the positively charged amino acids, which changes the conformation of the protein: A membrane pore is formed from the hydrophobic stretch of amino acids between S5 and S6. The voltage-sensing domain consists of this pore as well as the S1-S4 segments at the periphery. The S4 segments sense depolarization and move the voltage-sensing domain within the lipid bilayer where it latches onto the pore domain of the next channel. This binding causes the pore domain to undergo a conformational change to open the pore so ions can flow through.

With regard to neurons, what is meant by excitatory or inhibitory inputs?

Excitatory inputs induce the generation of an action potential in the postsynaptic neuron while inhibitory inputs block the generation of an action potential.

Botulinum toxin (Botox) binds to high affinity recognition sites on neuromuscular cholinergic nerve terminals. Demonstrate your knowledge of neurotransmission at the NMJ by describing the molecular steps that begin with the action potential reaching the motor neuron axon terminal and end in muscle contraction. Use this knowledge to describe the molecular mechanisms underlying the effects of Botox on wrinkles.

First the presynaptic membrane is depolarized and the voltage-gated calcium channels open allowing calcium to flow into the presynaptic cell. The SNARE proteins facilitate vesicle fusion and neurotransmitter release into the synapse, allowing NTs to bind to the postsynaptic membrane receptors, depolarize the muscle cell, and it will contract. Botulinum toxin cleaves the SNARE proteins so this prevents vesicles from being brought closer to the presynaptic membrane and decreases vesicle fusion with the membrane. Plus, if botox cleaves synaptotagmin on the vesicle membrane, than its calcium sensing properties won't work and vesicle fusion would not be triggered. It also looks like when botulinum toxin binds to the high affinity recognition sites on the presynaptic terminals, it prevents, or at least decreases, normal acetylcholine release from the presynaptic terminal into the synaptic cleft. Thus, acetylcholine cannot cross the synaptic cleft and binds to acetylcholine receptors on the postsynaptic receptor and trigger depolarization to cause muscle contraction. All of these actions make vesicle fusion and neurotransmitter release unlikely and prevent/decrease depolarization in the postsynaptic target. If depolarization does not occur, then muscle contraction will not occur. Muscle contraction in facial muscles over a long period of time leads to wrinkles, so if this muscle contraction can be prevented, then botox administration can lead to decreased wrinkles.

What would happen if you lesioned the left LGN? Why?

If you lesioned the left LGN, you would lose vision of the right visual field because the left LGN receives input from the right half of the visual field.

What experimental evidence supported the neuron doctrine?

GOLGI STAIN later on: In vitro axon growth experiments via tissue culture technologies showed that individual neurons begin with cell bodies and then axons will grow out, but they will not fuse with other neurons. Electron microscopy also provided experimental evidence in support of the neuron doctrine by visualizing the synaptic cleft, once again showing that neurons do not fuse together to form a continuous reticular network, but instead have this gap between neurons where communication takes place.

What are GRN and CRMS? Describe their role in retinogenesis.

GRNs refer to gene regulatory networks which are essential for retinal development. They establish spatial, temporal and cellular context specific controlled changes in gene expression patterns along with transcription factors and by influencing CRMs - all to acquire and maintain a specific cell type identity. CRMs are cis-regulatory modules consisting of transcription factor binding sites on a DNA strand - can promote or inhibit transcription. They are acted upon by GRNs to promote retinogenesis.

How could Golgi and Cajal both be looking at the same preparation, yet come to such different conclusions about the neural circuitry?

Golgi made a biased interpretation based on his stain that matched with the currently accepted reticular network theory of his time that probably aligned with his previous training instead of making an unbiased and objective interpretation of the evidence he collected himself. Cajal used the same evidence, the Golgi stain, to make a new conclusion that individual neurons make up the nervous system, and thus, refuting the reticular network theory.

Why are men more likely to be red/green color blind?

Green-red color blindness mutations have been mapped to the opsin gene on the X chromosome with M and L linked on this chromosome. Since males only have one X chromosome while females have 2, a male will more likely be affected by green-red color blindness because he only needs to inherit one "bad" copy.

From psychophysical studies it was determined that each rod reports the absorption of a single photon. What were the data and reasoning behind this conclusion?

Hecht and colleagues had subjects sit in a dark room for at least 30 minutes prior to any experiments. This was done to allow for detection of the lowest amount of light input. Then lights were shined on the peripheral area dense with rods and 1 ms episodes of 510 nm light exposure (rods are most sensitive to this wavelength). The number of photons in each flash was varied and the subjects would report if they saw light or not. The resulting data quantified the frequency of light flashes seen plotted against relative light intensity, showing that 5-7 photon absorptions must occur in a retinal field of 500 rods for the person to see the light flash. Thus, each rod likely reports the absorption of a single photon because it would be unlikely that one rod would absorb multiple photons.

The attached figure shows an I-V plot for the NMDA receptor in the presence of external Mg2+. What would the curve look like if Mg2+ were removed from the extracellular media and why?

If Mg2+ is removed from the extracellular media, the curve would appear nearly linear. As shown, at negative potentials, Mg2+ blocks the NMDA receptor from opening, while depolarization (less negative potentials) removes the Mg2+ block and the channel can open. Thus, the curve at negative potentials would resemble the curve at positive potentials in the absence of Mg2+ because the NMDA receptor conductance is unchanging and only depends on ligand binding (voltage sensitive, not voltage-gated).

In the absence of NE application, the inability of Gα to hydrolyze GTP:

If the G-protein cannot return to its inactive resting state, then the signaling pathway cannot be terminated and the signaling cascade will continue. Phosphorylation of VGCCs would keep occurring, thus increasing the open probability of calcium channels.

According to the Goldman-Hodgkin-Katz equation, if the membrane were more permeable to Na+ at rest, instead of K+, what would the approximate resting membrane potential be? Why?

If the membrane were more permeable to Na+ at rest, the resting potential would resemble the equilibrium potential of Na+. This is because the equation shows that each ion makes an independent contribution and Na+ ions would be flowing into the cell along the electrochemical gradient (from high to low concentration, from more positive to less positive), making the intracellular environment more positive. If the permeability of Na+ increases, the contribution of Na to the resting potential increases as well (and K+ and Cl- remain smaller), thus the total resting potential becomes more positive to resemble the equilibrium potential of Na around +50 - +58 mV.

What is the relationship between the cell cycle and interkinetic nuclear migration during cell proliferation in the neural tube.

In G1 of the cell cycle, the nucleus of the precursor cell is near the ventricular surface. In stage S, the nucleus migrates towards the pial surface and DNA is replicated. In G2, the nucleus migrates back to the ventricular surface with two copies of the DNA. In M phase, the nucleus remains next to the ventricular surface and the cell tracts its arm from the pial surface and then divides to form two cells. The cycle can then repeat or the cell can be arrested/postmitotic.

In figure below, (A) shows the dark-adapted response to a flash of light in wild type and GCAP knockout mice, and (B) shows the rod response to a flash of light relative to the same flash after dark adaptation. Based on these data, what is one mechanism of adaptation?

In the GCAP knockout, increased light intensity will still cause the decrease in calcium levels as CNG channels close. However, because GCAP is absent, GC activity will not increase, and more cGMP will not be produced. Thus, membrane depolarization will not occur. This means more light will not be needed to get the same amount of hyperpolarization, hence why the curve in B is shifted left. However, for the wild-type with GCAP, more light will be needed to get the same response, hence the curve is shifted right. We also see that the wild-type has faster light responses which allows for fast recovery as we see in A when compared to the knockout which cannot adapt as well.

Cell division in the early neural tube is symmetrical, with a vertical cleavage plane. Explain why.

In the early developmental stage, symmetric division occurs with a vertical cleavage plane to increase the pool of neural precursors. The vertical cleavage plane keeps both daughter cells in the ventricular zone instead of one migrating away as well as gives both daughter cells equal distribution of apical and basal cell components, so this allows both to undergo division again and keep increasing the number of daughter cells.

Retinitis Pigmentosa is a degenerative disease of the retina in which mutations cause the progressive death of rod photoreceptors in early adulthood to mid-life. Eventually the death of the rods leads to oxidative damage and death of cones which occurs after almost all the rod cells have died. Based on your knowledge of the distribution of rods and cones and how they function in the retina, describe what types of visual losses the patient would experience from early to late stages of the disease? Explain your reasoning.

In the early stage of the disease, the patient would begin to experience visual losses in the periphery of their vision. This is because rods are concentrated peripheral to the fovea, which is at the center of the retina, and the death of the rods occurs first in this disease. Rods are also responsible for vision in low light conditions, so a patient's ability to see well in lower intensity light (like at night, in the dark) would begin to diminish as the rods die and sensitivity is lost. In the late stage of the disease, the patient would begin to experience visual losses in the center of their vision. This is because cones are concentrated in the central retina, aka the fovea, and the death of the cones occurs following the death of the rods. Cones are also responsible for high-acuity and color vision, so a patient's ability to see colors and distinguish details of objects would diminish, eventually leading to blindness.

If the surround of the light source is now changed from being completely black to gray, how would the response of the bipolar cell change? Would it be more hyperpolarized or more depolarized than in the previous question? Explain your answer.

In this situation, the bipolar cell would become more hyperpolarized in response to light as the center photoreceptor becomes more depolarized and releases more glutamate than before. The glutamate will bind to the metabotropic receptors and inhibit the bipolar cell from releasing glutamate, so it is less excited.

What is the probable molecular mechanism of reduced light adaptation involving GCAP?

Increased background light intensity causes some cGMP-gated cation channels to close, meaning intracellular calcium decreases. This causes GCAP (guanylate cyclase activating protein) activity to increase, activating guanylate cyclase, meaning more cGMP is produced. CGMP can bind to the cation channels and allow membrane depolarization to occur. This makes phototransduction less efficient, meaning more intense light is needed to more strongly activate phosphodiesterase to reduce cGMP levels. Thus, more light is needed to make sure the same amount of hyperpolarization occurs.

1. In figure above, what do you predict would happen if you increased the duration of stimulus 4?

Increasing the stimulus duration does not change the duration or amplitude of the action potential but it will create multiple spikes of equal size and shape - more frequent spikes occur with a sustained suprathreshold stimulus than a sustained normal threshold. The Na channels close and K+ open so hyperpolarization phase also becomes faster, allowing the cycle to repeat and Na+ channels are reactivated.

Based on the known cortical circuitry and the response of complex cells, in which cortical layer is it likely you would find complex cells?

Information flows from layer 4 where simple cells receive input from LGN neurons and then to layers 2/3 and then layers 5/6 of the visual cortex as complex cells receive input from simple cells, so complex cells are usually present in layers 2/3 and 5/6.

How is information is transmitted between neurons?

Interneuronal communication at electrical synapses consists of direct transmission of membrane potential changes via ions flowing across gap junctions between adjacent neurons. Interneuronal communication at chemical synapses consists of an action or graded potential triggering neurotransmitter release into the synaptic cleft. The neurotransmitters then diffuse to and bind the receptor terminals of the postsynaptic neuron where the next potential is induced.

The figure below shows the spectral sensitivity of human cones. What three wavelengths do we respond best to? What cones would respond to a very bright light whose wavelength was between 450 nm and 500 nm?

It appears that we respond best to wavelengths of 450 nm, 550 nm, and 575 nm which we can see at the three peaks. From the figure, all three types of cones would respond to a light whose wavelength was between 450 and 500 nm because all three cone subtypes overlap at this point in this graph.

You identify a new neuron in the Drosophila brain and find it contains acetylcholine. Based on this identification this neuron would you expect it to be excitatory? inhibitory? modulatory? Explain your answer.

It depends on the cell the neuron is connected to, so we cannot determine identification without more information. ACh can bind to both ionotropic and metabotropic receptors. Could be excitatory if nicotinic (ionotropic) receptor, excitatory/inhibitory if muscarinic (metabotropic) receptor, or neuromodulator depending on location of receptors on target cell and downstream effectors.

graded potential

transmits information throughout the nervous system but depends on membrane potentials that vary in magnitude depending on stimuli strength and receptor neuron sensitivity. These potentials can be triggered by sensory stimuli and neurotransmitter release

What would happen to the voltage across the membrane if you blocked phosphodiesterase (PDE) and then stimulated the photoreceptor with light? Why?

Light normally activates phosphodiesterase which then decreases cGMP levels to trigger membrane hyperpolarization. Inhibition of phosphodiesterase would not lead to these changes, so the membrane polarization would stay the same if the photoreceptor is still stimulated with light.

In photoreceptors, in the presence of light, would the injection of cGMP depolarize, hyperpolarize or have no effect on the voltage across the membrane? Why?

Light normally triggers a reduction in cGMP and then activation of phosphodiesterase catalyzes hydrolysis of cGMP to GMP. However, an injection of cGMP would increase conductance of the cGMP-gated cation channel and cause rod depolarization, mimicking dark conditions. Increased concentrations of intracellular cGMP also inactivate phosphodiesterase activity that normally leads to hyperpolarization.

In the vertebrate nervous system, neurotrophins play important roles in development and maintenance. Describe how neurotrophin binding to Trk receptors can regulate gene expression.

Neurotrophins bind to Trk receptors and a neurotrophin dimer brings two Trk receptors close together. The kinase domain of one Trk receptors phosphorylates tyrosine residues of the second receptor. The phosphorylated tyrosine residues are now binding sites for adaptor proteins which will initiate downstream signaling. One adaptor is Shc which becomes phosphorylated by Trk after binding. Then Grb2 and Sos proteins are recruited to activate Ras. Activated Ras-GTP binds to Raf which can then activate Mek which then activates Erk. Lastly, Erk activates transcription factors to promote gene expression related to neuronal survival and differentiation

Using the diagram below as a reference, describe the signal transduction pathway that begins with acetylcholine binding to a metabotropic acetylcholine receptor including all the steps represented on the diagram. What would happen to the downstream substrates of this signaling pathway in the presence of an IP3 receptor inhibitor. Explain your reasoning.

Once acetylcholine binds to the metabotropic acetylcholine receptor on the extracellular side, this GPCR undergoes a conformational change in its transmembrane portion that lets the cytoplasmic domain bind with the trimeric G-protein. This binding catalyzes the release of GDP from the inactive alpha subunit, allowing GTP to bind in its place. GTP binding dissociates the alpha subunit from the beta-gamma subunit. In this case, the alpha subunit is Gq and it then goes to bind and activate phospholipase C (PLC). Then PLC cleaves PIP2 to form IP3 and DAG. IP3 then goes to bind to an IP3-gated calcium channel on the endoplasmic reticulum membrane, which releases calcium into the cytosol. On the other hand, DAG remains associated with the plasma membrane and activates protein kinase C (PKC). The calcium released from the ER into the cytosol binds to both PKC and calmodulin. PKC goes on to phosphorylate downstream targets. Calmodulin can also phosphorylate many pathways, but in this figure calmodulin is activating CaM kinase II which can then act on other targets as well. In the presence of an IP3 inhibitor, IP3 would be unable to bind to the IP3-gated calcium channel on the ER membrane, so calcium would not be released into the cytosol. Thus, calcium would not bind to PKC and calmodulin and their downstream targets would not be activated.

Explain the formation of an RA gradient in the hindbrain and describe how it contributes to patterning the CNS.

RA is a metabolite of vitamin A and RA signaling is required to establish the correct patterning of the hindbrain. Retinol enters the cell and is metabolized to RA which crosses the nuclear membrane and binds to nuclear receptors RXR and RAR. These nuclear receptors also bind to the DNA and act as transcription factors to affect gene expression. One such target is the hox genes. There are also specific genes that code for enzymes that are important in producing and degrading RA. The enzyme raldh2 helps produce RA in the caudal hindbrain while the enzyme cyp26 helps degrade RA in the anterior hindbrain. With the simultaneous production and degradation occurring, a RA gradient is established with higher concentrations of RA near the caudal hindbrain and lower concentrations in the anterior hindbrain. This gradient is necessary for hindbrain segmentation with the 7 rhombomeres. If there is too much RA present, then the focus will be on posterior structures and anterior structures will be missing. If there is too little RA to be found, then the focus will be on anterior structures and posterior structures will be missing.

Gain of function experiments

try to figure out if an added component to a system is sufficient for the system to perform a specific function. For example, gain of function experiments with epileptic patients showed that electrical stimulation/activation of specific motor cortical neurons was enough to produce specific muscle twitches.

What happens to the organization of that image from the retina to the LGN and then to primary visual cortex? That is, what is the anatomical organization of the visual circuitry? Include the organization/location of the terminal projections in the LGN and cortex.

Since the pumpkin appears in the right half of the visual field, the temporal projection of the left eye and nasal projection of the right eye would project to similar regions in the left LGN because they are looking at a similar point in the visual field. The projections would be in separate LGN layers because they are not coming from the same eye. The temporal projection (ipsilateral) would project to layer 2, 3 or 5 and the nasal projection (contralateral) would project to layer 1, 4 or 6. Then the left LGN neurons would project to layer 4 of the visual cortex, again in neighboring regions but not overlapping because the information remains segregated until the next V1 layer.

A series of critical experiments showed the properties of chemical synaptic transmission at the neuromuscular junction. In Fig. 1. above, voltage across the membrane was recorded with an intracellular electrode. The investigators applied ACh to the muscle via iontophoresis in the presence of TTX. Why did they use TTX?

TTX binds to voltage-gated sodium channels and prevents them from opening and allowing sodium ions to pass out of the cell to generate an action potential. If no action potential is generated, then muscle EPPs do not occur, hence there is no response from the muscle fiber. However, when ACh iontophoresis was used in the presence of TTX, action potentials and muscle EPPs did occur. This allowed them to investigate the role of ACh in neurotransmission - removed influence of motor neuron to see if ACh could stimulate muscle EPP.

There are two main types of integration of signals, what are these and what is the difference between the two?

Spatial integration - excitatory postsynaptic currents, from synapses at different locations that are activated at the same time, are added together when they converge along the same path to the cell body Temporal integration - excitatory postsynaptic currents from synapses that are activated within a specific amount of time are added together

Connectivity through electrical synapses comes close to the idea of the nervous system espoused by the proponents of the Reticular Theory. Discuss the structure of electrical synapses and how different aspects of electrical synapses may conform to the Reticular Theory or the Neuron Doctrine.

Structure: connexin proteins contribute to gap junctions; hemichannels form pores Paired recording experiments showed that networks of electrically coupled cells can act as detectors for synchronous activity, and a network of cells working together sounds similar to the reticular theory's nerve network. Electrical synapses are also connected via gap junctions, which lends itself to the reticular theory as the cells are physically connected and moving small peptides instead of the chemical synapses being physically separated which fits more with the neuron doctrine. However, the cells do not completely share their contents as lipids, nucleic acids, carbohydrates, and bigger proteins cannot pass through gap junctions. Signals can also flow in both directions (presynaptic to postsynaptic and vice versa) which does not conform to the neuron doctrine which was more focused on the functional polarity of neurons (presynaptic to postsynaptic only) - DID NOT talk about

What causes synaptic facilitation? Does it represent a long-term change in synaptic strength?

Successive action potentials trigger progressively larger postsynaptic responses which could be due to an initial low release probability, accumulation of calcium in the active zone, or from closely spaced stimuli. This represents a short-term change in synaptic strength. - Successive action potentials close together

What causes synaptic depression? Does it represent a long-term change in synaptic strength?

Successive action potentials trigger progressively smaller postsynaptic responses which could be due to initial high release probability, larger release of neurotransmitters with the first stimuli then a smaller release with subsequent stimuli, or the pool of ready releasable vesicles becomes exhausted with closely spaced stimuli (no chance to get replenished). This represents a short-term change in synaptic strength.

What is meant by symmetric and asymmetric cell division? How do they contribute to proliferation and differentiation of retinal progenitor cells?

Symmetric cell division refers to the production of two daughter cells that are similar whereas asymmetric cell division produces two daughter cells, one of which is similar to the original cell and the other is differentiated. Symmetric division contributes to the proliferation of retinal progenitor cells by simply increasing the number of progenitor cells. It also contributes to differentiation by producing two postmitotic terminally differentiated daughter cells later in development. Asymmetric division contributes to both proliferation and differentiation by increasing the number of retinal progenitor cell pool by one daughter cell each time and producing one terminally differentiated postmitotic daughter cell. -affected by apical and basal polarity

1. The brains of patients with Alzheimer's disease show degradation of microtubule function, in part from over-phosphorylation of the microtubule-associated protein, tau. What might happen to neurons when microtubule function is disrupted?

Tau appears to play a role in stabilizing microtubules and allowing for microtubule assembly. If over-phosphorylation of tau occurs, tau may dissociate from the microtubule and aggregate with other tau proteins to form neurofibrillary tangles. This dissociation of tau would lead to destabilization and disassembly of microtubules. The plus end of the microtubule could be degraded and shortened. If this is true, transport from the center of the cell to the periphery (including dendrites and axons) would be inhibited or made more difficult (slowed down). The motor proteins (dynein, kinesins) that use microtubules to transport cargo to the periphery would not be able to reach the center of axons and dendrites. Since microtubules allow for long-distance transport, over-phosphorylation of tau will inhibit long-distance transport in neurons, thus, necessary cargo would not be transported. These neurons will die after prolonged disruption or neuronal transport.

In the cochlea of the ear, sounds waves are turned into electrical signals through special cells called hair cells. When a wave travels through the cochlea it moves the 'hairs' and opens ion channels that are permeable to K+ and Ca2+. These 'hairs' are in a fluid that has a very high K+ concentration compared to the inside of the cell. When the ion channels open, in what direction do K+ ions flow and why? Based on this relative concentration difference of K+, what do you predict the equilibrium potential for K+ will be: positive, negative or zero?

The K+ ions will flow down the chemical gradient which is from the extracellular fluid and into the inside of the cell. As the K+ ions flow inward, the extracellular environment becomes more negatively charged, increasing the electric potential across the membrane and slowing down further movement of K+ ions into the cell against the electrical gradient. Eventually the equilibrium potential is reached where there is no net flow of K+ ions - this potential will be positive (according to the Nernst equation) - intracellular membrane potential would be positive compared to extracellular environment. - More positive K+ outside, so positive potential inside would be needed to offset this.

Why is the NMDA receptor a good coincidence detector?

The NMDA receptor is a good coincidence detector because it only opens after both presynaptic glutamate release and postsynaptic depolarization occurs. Coincidence = activation of presynaptic cell and postsynaptic cell

If the surround of the light source is now changed from being completely black to gray, would the retinal ganglion cell produce more action potentials or fewer action potentials than in the previous question? Explain your answer.

The ON-center/OFF-surround retinal ganglion cell would produce fewer action potentials as firing of action potentials decreases as the diameter of the light spot increases. This is because the ON bipolar cell is less excited and releasing less glutamate. Thus, the RGC is less excited and will not fire as many potentials.

In the absence of application of NE, intracellular addition of cAMP:

The addition of cAMP would still cause the cAMP phosphorylation cascade even without binding of NE, so phosphorylation of VGCCs would still occur, increasing their open probability.

What is the basic organization of the hydra's nervous system? Describe how this system of organization fits with the way in which the hydra encounters its environment?

The basic organization of the hydra's nervous system is referred to as a nerve net made up of a double layer arrangement of sensory and motor neurons distributed diffusely throughout the body wall of the hydra which makes sense for the mainly symmetric shape of the hydra's body. There are some areas of centralization around the hydra's mouth and at the base of the tentacles where aggregation of neurons occurs. These areas of centralization appear to be related to the coordinated movements of ingestion as the tentacles can sense food and bring it to their mouth as well as related to the general coordination of the tentacles needed for locomotor behavior.

You are out in the middle of the desert star-gazing trying to see the North Star. What is the best way to look at the star? With the fovea or from the side of your visual field? Why?

The best way to look at the star is to look for the North Star from the side of your visual field. Since you are stargazing at night, high-acuity color vision is not necessary and you are trying to see in dark light, thus, retinal areas enriched in rods will be most appropriate for this scenario where greater sensitivity is required.

If the surround of the light source is now changed from being completely black to gray, how would the center photoreceptor respond? Would it be more hyperpolarized or more depolarized than in the previous question? Explain your answer.

The center photoreceptor is still hyperpolarized but the response would become more depolarized than the previous scenario as there would be less lateral inhibition occurring since the surround photoreceptors are not activating the horizontal cell. It now releases slightly more glutamate than before.

Using the voltage clamp technique, Hodgkin and Huxley found an early inward current and a later outer current (Figure 1, recorded current). What would happen to the current if you would apply tetrodotoxin to the bath before changing the voltage across the membrane? Please select between currents A and B and explain your answer.

The early inward current of the recorded current in figure 1 is caused by the influx of Na+ into the cell while the later outer current is caused by the efflux of K+ out of the cell. Tetrodotoxin binds to voltage-gated sodium channels and blocks the passage of sodium ions into the cell. Thus, if tetrodotoxin is added to the extracellular environment, it would not allow sodium ions to pass into the cell and the early inward current will not occur. However, potassium ions are not affected so the later outer current is still allowed to occur - this scenario resembles line A.

In Fig.2 above, in the top left trace there are three motor nerve stimulations that occurred at the arrow. Why is the flat trace flat and, based on what we know now, what is the reason that the other two traces are multiples of each other?

The flat trace is flat because no EPP occurred and there was no evoked response - a fail. In the second trace, the smaller depolarization is caused by the release of a single quantum (packet). In the third trace, the larger depolarization is caused by the release of two quanta, a multiple of the single quantum. Each vesicle has a similar number of neurotransmitters so that's why multiples occur. This suggests that stimulation causes the coordinated release of packets of neurotransmitters.

Flatworms do not have nerve nets. How is their nervous system generally organized? Describe how this system of organization fits with the way in which flatworms encounter their environment?

The flatworm nervous system consists of ganglia clusters connected by longitudinal and transverse nerve cords and this reflects a centralization of the system. This setup also caters to the bilateral symmetry of the flatworm. The cephalic ganglia are located by the flatworm head and their larger size and more complex nature demonstrates their importance to the localization near the head as this is where special sensory receptors are located so this cephalization helps the flatworm swim forward rapidly.

Describe how retinal structure differs at the fovea compared to the rest of the retina.

The fovea is located at the center of the retina, and this region is highly concentrated with cones (makes up 1% of the total retinal area). The retinal areas next to the fovea are highly concentrated with rods. The fovea is also devoid of blood vessels and has displaced retinal layers (thin) for the direct path of light.

What is the function of endocytosis? Describe an important role for endocytosis in neuronal signaling?

The function of endocytosis is to take up extracellular components or transmembrane proteins and bring them into the cell (then to lysosomes or late endosomes). Endocytosis plays an important role in neuronal signaling as it regulates the number of receptors on the postsynaptic neuron that interact with the released neurotransmitters in the synaptic cleft. Also reuptake of neurotransmitters via endocytosis after binding to receptors on presynaptic cell.

What structures of the eye control the amount of light entering the eye and focus the light onto the retina? From what part of the developing optic tissue are they derived?

The iris controls the amount of light entering the eye. The lens focuses the light onto the retina. Both structures arise from the ectoderm - the iris is derived from the anterior rim of the optic cup and the lens is derived from the surface ectodermal cells that lie over the optic vesicle (lens placode). Optic vesicles come from the diencephalon (of the forebrain).

In the figure above, stimulus 4 is of larger amplitude than stimulus 3. What happened to the action potential when a larger stimulus was provided and why?

The magnitude of the action potential did not change with the larger magnitude of stimulus 4 because the action potential threshold is reached in both scenarios, but it did fire faster. The greater stimulus (more current) caused faster depolarization because there are more Na+ voltage-gated channels open = more Na+ flowing into the cell faster.

How has our understanding of segmentation and hox gene patterning in fly embryos contributed to our understanding of vertebrate brain development?

The molecular basis of segmentation was first discovered in fruit flies when homeobox genes were identified. Homeotic genes code for transcription factors that bind to the homeobox sequence of DNA and modulate gene expression. Thus gene expression leads to distinctive segmentation of the embryo and different identities among neuronal types in humans. The fly embryo differentiated into segments leading to the head, thorax and abdomen. So if there was a mutation associated with these genes, then there was a disruption in a specific morphological feature. Similar hox genes were discovered in humans that also contribute to segmentation but in the vertebrate nervous system, leading to fully differentiated brain regions.

In the presence of NE application, the inability of GDP to dissociate from Gα:

The open probability of calcium channels would not change because the G-protein cannot be activated even in the presence of NE as GDP cannot be exchanged for GTP, so the signaling pathway would not occur and calcium channels would not be phosphorylated.

What are the structures derived from the posterior optic cup and what are their functions?

The pigmented and neural retina is derived from the posterior rim of the optic cup. The retinal pigment epithelium is derived from the outer layer of the posterior optic cup while the multilayered neural retina is derived from the single inner layer of the posterior optic cup. The retinal pigment epithelium structurally supports the retina and enables photoreceptor cells to detect light (phototransduction; epithelial transport, phagocytosis - cleans up waste from photoreceptors (leads to macular degeneration if this is lost) - and secretion). The retina codes for visual information beginning with photoreceptors and ending in the brain to form the final image like we have previously discussed.

Lewis Wolpert famously said, "It is not birth, marriage, or death, but gastrulation, which is truly the most important time in your life". What are the primary consequences of gastrulation and how do they contribute to the future establishment of neural tissue?

The primary consequences of gastrulation include the formation of the (1) primary germ layers, (2) notochord, and (3) primary body axes. From the bilayer structure, cells from the prospective ectoderm will dive down to form the notochord, some remain between the two layers to form mesoderm, and some dive down and displace bottom hypoblast layer to form the endoderm. The notochord will define the embryonic midline and is the major axis of symmetry for the body which determines the position of the nervous system and is required for neural differentiation. The neuroectoderm, above the notochord, gives rise to a population of neural precursors that have the ability to become neurons, astrocytes, and oligodendrocytes; thus, giving rise to the entire nervous system. (2) Signaling forms the dorsal-ventral and anterior-posterior axes.

What kind of visual features can neurons in V1 respond to? Why?

The primary visual cortex contains two types of cells, simple and complex. Simple cells are best stimulated by bars of light in a specific orientation, thus responding to lines and edges of specific regions. Complex cells also respond to bars of light on a dark background or dark bars on an illuminated background but are more orientation specific than simple cells and can be referred to as more abstract line and edge detectors over larger and more complex receptive fields. Some V1 cells are also direction selective to moving bars of light (probably complex cells). -V1 respond to lines, edges, and motion

What can you conclude about the plasticity in owl auditory maps from the experiments in which juvenile or adult barn owls are fitted with prisms that cause a mismatch between auditory and visual stimuli?

The prism experiments demonstrated that barn owls have neural plasticity which allows them to adapt to a changing environment. In this case, the changing environment was the addition of a prism over their eyes that caused the owls to adjust their auditory maps to match the altered visual map. This experiment suggested that the nervous system undergoes changes to respond to new experiences and improve learning abilities. The owls were also able to adjust their auditory maps back to normal after the prisms were removed. The experiment also showed that owls that reached sexual maturity prior to prism attachment did not have the plasticity to fix the mismatch between auditory and visual maps, suggesting that plasticity declines with age. Adult owls were able to overcome this barrier if they had previously experienced the prism induced visual shift as a juvenile and were readily able to adjust when placed in the same scenario or by adjusting to the prism in small increments instead of the one large 23 degree shift.

In vertebrate CNS neurons, synapses are on dendritic spines. Most of the postsynaptic receptors are on the spine head, which are attached to the dendrite through a very small spine neck. What is the consequence of this small neck?

The spine neck chemically and electrically compartmentalizes each synapse so that it can modulate the synapse semi-independently from other synapses at a localized to site. Signals from different synapses can then be merged post-modulation. Depending on the input neuron, the strength of individual synapses will vary and can be modified by the activity of the previous activity of the synapse.

If the surround of the light source is now changed from being completely black to gray, how would the photoreceptors in the surround respond? Would they be more hyperpolarized or more depolarized than in the previous question? Explain your answer.

The surround photoreceptors would now become hyperpolarized since light is hyperpolarizing for photoreceptors. This means the horizontal cell will be less activated and the photoreceptors will be less inhibited. Thus, more glutamate will be released from both the center and surround photoreceptors than in the previous scenario. This will cause the ON bipolar to be less excited and there would be less firing from the RGCs.

Explain the advantages of the simple nerve net over the direct stimulation of effector cells observed in sponges in terms of information processing.

The two layer nerve net allows for increased response complexity and integration over the direct stimulation of effector cells we see with sponges because if a stimulus activates a single sensory neuron of the nerve net, the one sensory neuron will activate multiple motor neurons. Then each motor neuron can activate multiple effector cells as well as other motor neurons to produce a large cascade from a single sensory neuron and stimulus interaction. On the other hand, sponges lack a nervous system, so there is only a direct one on one interaction between a stimulus and the effector cell and no information cascade can occur.

The nervous system consists of mainly two classes of cells, what are they and what is their functional role in the nervous system?

The two main classes of cells in the nervous system are neurons and glia. Neurons function to transmit signals over long distances throughout the nervous system while glia cells have more of a supporting role, such as myelinating axons (oligodendrocytes, Schwann cells), developing/regulating communication (astrocytes), and immune activity (microglia).

In order to understand how currents move during an action potential, Hodgkin and Huxley used the voltage clamp technique. Why was this technique so important for their discoveries?

The voltage clamp technique simplified measuring ion flow in response to changes in voltage without affecting the permeability of the ions. They were able to mimic an action potential using depolarizing voltages so that they could observe where individual ions were flowing during the action potential. By changing voltages and measuring currents, they were able to determine the conductances of sodium and potassium at different membrane potentials, thus linking changes in conductance with depolarization and movement of ions into and out of the cell. - how ion channels function overall, verified ion channels existed

What molecular mechanism contributes to the alignment between the presynaptic active zone and the postsynaptic density?

There are synaptic adhesion molecules that link the presynaptic active zone to the postsynaptic density. Neurexins of the presynaptic membrane bind to neuroligins of the postsynaptic membrane (heterophilic) and cadherins, present on both membranes, bind to each other (homophilic). There are other cell-adhesion molecules that align the active zone with the postsynaptic density.

What type of bipolar cell is illustrated (ON- or OFF-) and how does neurotransmitter release from the presynaptic photoreceptor result in the graded potential response in this cell?

This is an ON-bipolar cell. When light hits the center presynaptic photoreceptor, it is hyperpolarized while the surround is depolarized, so there is low glutamate release onto the ON bipolar cell. This means less glutamate binds to the metabotropic glutamate receptors on the bipolar cell to indirectly inhibit cation channels, and the ON bipolar cell is strongly excited. Thus, the ON- bipolar cell becomes depolarized in response to light and releases more glutamate to activate the RGC. The RGC then fires more graded potentials.

receptor potential

type of graded potential triggered by sensory stimuli which occurs at peripheral endings of sensory neurons

What have transplantation experiments revealed about cortical neuronal specification?

Transplantation experiments showed that cortical neurons adopt their cell fate after exiting the cell cycle, and their destination depends on the timing of this exit. Post-mitotic "early born" cells occupy deep layers while post-mitotic "later born" cells occupy more superficial layers. Transplantation experiments also showed that the earliest-born neurons in the subplate layer contain the instructions for the assembly of the rest of the cortex. -shows that fate is determined intrinsically prior to migration -fate isn't completely determined until the cell exits the mitotic cycle -cell fate becomes more restricted through rounds and rounds of division, so it loses capacity to give rise to cells that would remain in the deeper layer.

What would the projection pattern look like if you traced the path from two neighboring RGC from the left eye to the LGN and cortex?

Two neighboring RGCs in the left temporal retina would project to a similar region of the left LGN. Because the projections are coming from the same eye, the projections will also be in the same LGN layer. Then the LGN projections would project to adjacent, possibly overlapping, regions of layer 4 in the visual cortex. All of these axon projections are organized topologically, meaning RGCs adjacent to one another project to LGN neurons that are adjacent to one another and so on for LGN to V1. This allows spatial information in the retina to be correctly represented in the brain.

In experiment 2, what happened to the potassium channel activity when serotonin was added to the outside of the cell and cAMP was added to the inside of the cell?

When you add serotonin extracellularly, the same signaling pathway as mentioned above occurs, so the potassium channels are closing - we see only 2 channels open in the 5-HT trace while we see 5 open channels for the control. The same thing occurs for the cAMP addition where the potassium channels close and only 1 is left open while the 5 channels remain open for the control.

The figure below shows the currents from a rod and a cone in response to increasing magnitude of light flashes. How do these responses contribute to the higher sensitivity in low light and higher acuity in daylight conditions?

We can see that rods are more sensitive than cones with the bottom level of light intensity about 65 times lower for the rod to produce the same current - and rods operate in low light scenarios detecting small changes. While cones are less sensitive than rods, they can adapt to a broader range of light intensities, which are applicable during daylight conditions, and they can recover much faster allowing for faster reaction times to repeated stimuli which allows for high acuity vision.

1. Syt1 point mutation is a point mutation in synaptotagmin-1. How did the experiment shown in the figure above show that synaptotagmin was probably the sensor for Ca2+influx through voltage gated calcium channels in the axon terminus?

When endogenous synaptotagmin-1 is replaced with the point mutation version, calcium binding is reduced by 50%. There was a subsequent reduction of sensitivity of neurotransmitter release by 50% compared to the wild-type response which we can see with the lower slope, meaning more calcium is needed to get the same response as the wild-type. This suggested that synaptotagmin-1 is a sensor for calcium binding to cause vesicle fusion.

The investigators compared the evoked EPPs in low Ca2+/high Mg2+ saline to spontaneous EPPs (mEPP). What did they find (see Fig. 2, right) and what did they conclude?

When stimuli did trigger EPPs in the low calcium environment, the EPP amplitude was the same as the amplitude of the spontaneous mEPPs with a few EPPs two or three (rare) times the size. Reducing the calcium concentration even more decreased the frequency of EPPs but did not change the amplitude. The frequency of distributions of EPPs determined experimentally matched those calculated based on the Poisson distribution. This led to the conclusion that the mEPPs were the basic unit of synaptic transmission, thus normal EPPs were the result of hundreds of mEPPs together. They came up with the quantal hypothesis of neurotransmitter release which says that neurotransmitters are released in relatively uniform packets, with the evoked response in normal saline occurring from many packets being released at once.

The elicited response measured in muscle fibers after ACh iontophoresis was similar to that evoked by nerve stimulation. What did this tell the investigators?

Whether the response of the muscle fibers was provoked by ACh iontophoresis or motor nerve stimulation, they found that action potentials trigger release of ACh from the presynaptic terminals into the synaptic cleft, so a chemical intermediate is used rather than direct transfer of the action potential to the postsynaptic target.

In experiment 1, what happened to the action potential when cAMP and serotonin (5-HT) were added to the extracellular space of the neuron (top trace) and cAMP was injected intracellularly into the neuron (bottom trace)?

With the addition of 5-HT (serotonin) in the extracellular space, we see the action potential is prolonged in the top trace (slower rate of repolarization). This is because serotonin binds 5-HT receptors to increase cAMP production which activates PKA. PKA then phosphorylates potassium channels, decreasing the open probability of the channels. If less potassium channels are open, then K+ efflux decreases and the rate of repolarization back to the resting membrane potential is slower, thus the action potential duration is longer. NT release is also triggered. The addition of cAMP intracellularly causes this same amplification cascade to occur so the action potential is also prolonged in the bottom trace (slower rate of repolarization).

If you stimulate a glutamatergic presynaptic neuron and record the response in the postsynaptic neuron with NMDA receptors, will you get no response? a depolarizing response? a hyperpolarizing response? Why?

You will not get a response unless both glutamate and glycine are bound to the NMDA receptor, and the postsynaptic neuron is depolarized.

action potential

a depolarizing change in the membrane potential that causes an impulse to pass through the axon and thus transmit information throughout the nervous system

You take a 'normal' oocyte and apply ACh by iontophoresis to the oocyte and record the resulting current response with voltage clamp: (a) What will the resultant current be? Explain. (b) What would happen if you first inject the 'normal' oocyte with mRNAs for AChR subunits? Explain your answer.

a. A normal oocyte does not have the ability to respond to ACh release if it does not have any acetylcholine receptors, so there will be no resulting current b. If you inject mRNAs for all 4 AChR subunits, ACh release will now trigger a net inward current. If the oocyte has a negative resting membrane potential with more K+ inside and more Na+ outside the cell, then the driving force will produce more Na+ influx than K+ efflux. Now that the receptors are present, these cation channels can allow the flow of ions to occur. This causes a net inward current to depolarize the oocyte membrane. However, you do need all of the subunits to be present

Loss of function experiments

based on figuring out if a missing component of a system is necessary for its function. For example, the brain lesions in Wernicke's patients caused loss of language comprehension, so it can be concluded that Wernicke's area is necessary for language comprehension

correlation experiments

investigate whether there is a relationship between two variables, but cannot determine cause and effect relationships without manipulating variables in an experimental setting. For example, there is a more positive correlation between identical twins for intelligence quotients than between two random people as well as fraternal twins, meaning identical twins bear more resemblance.

synaptic potential

one type of graded potential is a synaptic potential which is triggered by neurotransmitter release by the presynaptic neuron