1. (15 pts.) You are to consider the following experimental evidence, answer the questions and then appropriately label the diagram below.

A. Systemic injection (like an intravenous injection) of picrotoxin ( a GABA_A receptor blocking agent) increased ACh levels in the caudate nucleus by 70%. Question: What could be the response to picrotoxin in cells receiving GABA projections? Explain: Since GABA is an inhibitory transmitter, blocking the action of GABA at GABA_A receptors will release inhibition onto cells. Receptive cells could increase their firing rates.

Question: What might increased levels of ACh indicate about striatal cholinergic neurons? A level of a transmitter reflects a balance between production and utilization. In the present situation, the increase in ACh level could reflect increased production, perhaps consequent to increased activity in cholinergic neurons.

B. Systemic injection of picrotoxin increased homovanillic acid (HVA, a metabolite of dopamine produced by MAO action on DA) levels in the caudate nucleus by 30%. Question: Where are the cells of origin of DA terminals in the caudate nucleus?

Cells in the substantia nigra send projections to the caudate.

Question: What might the increase in HVA indicate about activity of DA projections?

It could reflect increased activity in DA neurons. As activity increases, more DA would be released and more would be exposed to the action of MAO.

C. Direct injection of picrotoxin into the caudate nucleus did not change choline acetyltransferase (CAT) activity. Question: What does CAT do?

ChAT synthesizes the production of ACh from choline and acetyl-Co A.

Question: Compare these results of direct striatal injection of Picrotoxin to the effects of systemic injection indicated in part A. What does this comparison suggest about the site of action of Picrotoxin? These results suggest that the impact of systemic Picrotoxin on ACh levels in the caudate must not have been due to a direct action in the caudate.

D. Pretreating animals with an inhibitor of tyrosine hydroxylase completely prevented the effect of systemic picrotoxin injection on ACh levels in the caudate nucleus. Question: What is the function of tyrosine hydroxylase?

Ty-OH is the rate-limiting enzyme in the synthesis of the catecholamines such as DA.

NOW: Indicate on the diagram below which of the neurons use ACh, GABA or DA as transmitter. THEN indicate the region where picrotoxin is most likely exerting its effect.

The interneuron in the caudate is cholinergic (releases ACh as its transmitter)
The neuron projecting from the Substantia nigra to the caudate is dopaminergic.
The neuron projecting from the caudate to the substantia nigra is GABAergic.
Systemic Picrotoxin exerts its effect on caudate ACh by acting in the Substania nigra.

2. (12 pts.) Levels of cAMP were measured in slices of the cerebral cortex incubated under physiologic conditions (Basal values) or in the presence of different transmitter substances. The data are shown below.
* indicates significantly different from basal levels.

A. Outline or diagram the mechanisms via which neurotransmitters can influence cAMP levels in neurons.

Transmitters can bind to receptors that are linked to either Gs or Gi proteins. The G proteins when activated by transmitter binding to its receptor then either activate (Gs) or inhibit (Gi) adenylate cyclase, which converts ATP into cAMP.

B. From the data shown above, which transmitters added alone significantly altered cAMP levels relative to basal levels? Based upon the direction of the change, what specific type of receptor-effector mechanism is indicated?

VIP and NE both significantly increased cAMP levels implicating action at receptors linked to a Gs protein.

C. Different transmitters may elicit responses that converge onto a common cell signaling system. From the above data, which two transmitters given together affected cAMP levels in a manner that clearly indicates they are converging on a common mechanism? Explain

Combined exposure to VIP and NE produced an increase in cAMP levels that was greater than just the additive effect of their individual responses. This suggests that these two transmitters acting through their respective receptor systems converge on a common mechanism.

3. (5 pts.) Changes in mesolimbic DA projections have been implicated in addiction. The following table shows the effect of chronic exposure (14 days) to morphine or cocaine on the levels of total or dephosphorylated tyrosine hydroxylase (TH) in the nucleus accumbens. The data are given as percent untreated control values. '*' indicates a significant difference from control.

A. Where are the cells of origin of DA projections to the nucleus accumbens?

Cells in the ventral tegmental area (VTA) send projections to the nucleus accumbens.

B. Comparing the effect of chronic drug exposure on dephosphorylated TH vs. total TH would you predict that TH activity in the nucleus accumbens would be increased or decreased as a result of chronic exposure to the drugs. Explain

Chronic exposure to each of the drugs significantly increased dephosphorylated TH without changing total TH. Since the total amount of the enzyme did not change but the dephosphorylated portion did, the phosphorylated portion must have decreased. Since phosphorylated TH is the active form of the enzyme, the activity of TH must have decreased.

4. (12 pts.) The suprachiasmatic nucleus (SCN) of the hypothalamus receives a direct input from ganglion cells in the retina. Ganglion cells give rise to the optic nerve. This input synchronizes activity of the SCN "biologic clock" with the light-dark cycle. Light-induced phase-shifting of circadian rhythms are tightly correlated with light-induced expression of the immediate early gene, cfos in SCN cells. From the following information, you are to indicate mechanisms whereby light acting on the retina could induce cfos expression in SCN cells.

1. Glutamic acid applied to SCN induces cfos expression and the NMDA receptor antagonist, AP5, blocks the induction.
2. Glutamic acid increases the amount of phosphorylated CREB in SCN cells and AP5 blocks the effect.
3. Binding to CRE on DNA is enhanced by light.

Now, describe (or diagram) plausible mechanisms whereby light hitting the retina could induce cfos expression in SCN cells. Include information on the transmitter in the ganglion cell projection, the types of receptors and related mechanisms in SCN cells that would be critical, including intracellular messengers and enzymes that are suggested.

The evidence given suggests that the transmitter in the ganglion cell projection was glutamic acid (GLU) since application of GLU to the SCN mimicked the effect of light hitting the retinaÑand you remember that the transmitter in primary sensory neurons is thought to be GLU. The evidence also indicates that action of GLU at NMDA receptors is critical for the effect of GLU, since blocking the receptor prevented the response. But you know that for GLU to activate NMDA receptors and open the Ca++ channel, the membrane must be depolarized, so GLU probably also acted at AMPA receptors. The rest of the evidence showed that GLU acting at NMDA receptors increased the amount of phosphorylated CREB and that light increased binding to CRE on DNA. So now you have to connect GLU action at NMDA receptors to the effect on DNA. Ca++ entering through NMDA receptors could bind to calmodulin and the Ca/calmodulin complex would activate the respective kinase, which would enter the nucleus and phosphorylate CREB thereby enhancing transcription of the cfos gene.

5. (10 pts.) In an initial study, the levels of 5-HT and the activity of tryptophan hydroxylase were measured in the Locus Coeruleus and raphe nuclei of control rats and rats that had been subjected to an immobilization stressor for 150 min. per day for 7 days before analysis. The following results were obtained:

_____________________________________________________________________________
*Significantly different from control group, P<.05

To understand possible mechanisms mediating the change in TRY-OH activity following chronic immobilization, a recent study measured TRY-OH mRNA levels in raphe nuclei. The following results were obtained.

The data showed that 7X immobilization significantly increased TRY-OH in both the dorsal and median raphe nuclei.

a. Why measure TRY-OH mRNA in the raphe nuclei when TYR-OH activity was affected by chronic immobilization only in the locus coeruleus?

mRNA is a measure of gene transcription, which can only take place in cell bodies. The cell bodies of 5-HT terminals in the LC are in the raphe nuclei. Thus to detect an impact of the chronic stressor on gene transcription, determination must be made in cell body regions.

b. Provide a plausible mechanism that links the chronic immobilization-induced changes in TYR-OH activity in the locus coeruleus with the changes in TYR-OH mRNA levels observed in the raphe. What purpose did the change in enzyme activity in the LC serve (consider 5-HT levels in the different groups)?

Stressors induce release of 5-HT. Thus, repeated immobilization increased the utilization of 5-HT by neurons projecting to the LC. This would lead to an activation of enzymes of synthesis (TRY-OH in the LC. A second adaptive response to the increased demand for the transmitter appeared to be enzyme induction, the production of new protein in the cell body. The new protein would be sent to the terminal to assist in synthesis. The enhanced synthesis of 5-HT served to maintain levels of 5-HT constant in face of the increased demand. This demonstrates how adaptive mechanisms work to meet demands that threaten homeostasis.

6. (12 pts.) In the mature brain, GABA action at GABA_A receptors stabilizes the membrane potential near the resting membrane potential, thereby making it difficult for excitatory transmitters to activate cells. However, the following evidence suggests a different action of GABA in the immature brain. The first set of figures demonstrate the effect of application of the GABAA agonist ( mimicking agent), muscimol, on intracellular calcium concentration [Ca²⁺]i in immature vs. mature hippocampal neurons. This effect is compared to that resulting from application of the glutamic acid receptor agonist, kainic acid.

The data here showed that in immature neurons,the GABA agonist, muscimol, induced increased (Ca++)i, similar to the action of the glutamate receptor, kainate. GABA did not have the same effect in the mature neuron since muscimol did not increase (Ca++).

a. What do these data suggest that GABA acting at the GABAA receptor does to the membrane potential of immature vs. mature neurons? Explain

They suggest that in the immature neuron, GABA had a depolarizing effect, since voltage gated Ca++ channels (VGCC) open when the membrane is depolarized. In the mature neuron, GABA did not depolarize membranes.

The following data show the developmental changes in the chloride equilibrium potential at the GABAA receptor (left) and in the intracellular chloride concentration (right) in neurons.

These data showed that in the most immature neurons (E16-19), there is a high intracellular chloride concentration and that the resting membrane potential is considerably more negative than the Ecl (equilibrium potential for chloride). At P16, the intracellular chloride concentration has decreased and the Ecl and Em (resting membrane potential) are almost the same.

b. Based on the above data, which direction (into or out of the cell) would the chloride ion flow when GABA opens its receptor-linked chloride channel in neurons at E16-19 vs. at P16? (E is embryonic and P is postnatal).

At E16-19, when GABA opens the Cl- channel of the GABA_A receptor, Cl- will flow out of the cell due to the high intracellular concentration of the ion and the Ecl that is far less negative than the Em. At P16, very little Cl- will move because of the low intracellular concentration of the ion and the similarity between Ecl and Em.

c. How then could this data explain the age-dependent effect of GABA on intracellular calcium concentration?

At E16-19, as Cl- leaves the cell and moves the Em towards the Ecl, the membrane will depolarize. Depolarization will open VGCC and Ca++ will ente. At P16, since Ecl and Em are so close, little Cl- will move and the Em will not change very much. There will be no depolarization.

7. (12 pts.) Microdialysis probes were implanted in the locus coeruleus (LC) and prefrontal cortex (PFC) of conscious rats. Drugs were infused into LC and the effects of the drugs on NE released in the PFC were determined by sampling extracellular fluid in that region. The data are illustrated below; dashed line on figures indicates time over which drug was infused.

a. What is the relationship of NE terminals in the PFC to the LC?

Cell bodies in the LC send axon projections to the PFC

b. The fig. to the right illustrates the effect of infusion of tetrodotoxin (TTX), a sodium channel blocker, into the LC. TTX would prevent generation of action potentials. Why would this elicit a decrease in extracellular NE in the PFC?

The application of TTX to LC induced a decrease in release of NE from terminals in the PFC. TTX would prevent the generation of action potentials; hence there would be no propagation of action potentials down the axon, and no depolarization of the terminals in PFC. Release of NE depends on depolarization-induced influx of Ca++ . Without depolarization, there will be no release of transmitters from the terminals.

c. The fig. to the right illustrates the effect of infusion of clonidine, a noradrenergic alpha2 receptor agonist (NE mimicing agent), into the LC. Via what mechanism(s) could a NE receptor agonist in the LC induce a decrease in extracellular NE in the PFC comparable to that seen following TTX?

The application of clonidine to the LC led to a decrease in NE release in the PFC. Since clonidine is a receptor agonist, it would have mimicked the action of NE in the LC and activated the a2 receptors. If the a2 was an inhibitory autoreceptor, its activation would induce a decrease in the firing rate of LC neurons, which will be accompanied by a decrease in release of transmitter at nerve terminals. Autoreceptors on cell bodies regulate impulse generation.

8. (12 pts.) The amount of the enzyme tyrosine hydroxylase (TH) was measured in the locus coeruleus (LC) of rats using immunohistochemistry. In experiment 1, TH levels were measured following 40 min. of foot shock and 30 min. of high intensity noise and also in control animals not exposed to the stressors. One group of animals exposed to the stressors had been pretreated for 4 days with daily LC infusion of α-h-CRF, a specific corticotropin releasing Factor (CRF) receptor antagonist. In experiment 2, TH levels were measured after 5 days of chronic intraventricular infusion of CRF itself. The data from both experiments is shown below: *=significantly different from the control condition.

a. What substance is the primary neurotransmitter of cells in the LC? NE

b. What do these data implicate as an important factor in regulation of TH levels in response to environmental stressors? Explain

The stressor-induced increase in levels of TH in the LC appear to be mediated via action of CRF at specific CRF receptors. Treatment with a CRF receptor antagonist prevented the impact of the stressor on TH levels. Furthermore, application of CRF to the LC for 5 days in the absence of a stressor increased TH levels indicating that CRF can influence TH expression.

In experiment 3 the levels of TH were measured in the LC 24 hrs. following exposure to a high dose of the monoamine-depleting drug, reserpine. This drug prevents the uptake of monoamines into synaptic vesicles. One group of animals had been exposed to α-h-CRF for 3 days prior to reserpine treatment while the other group received vehicle (placebo). Control animals were not given the drug. The black and white bars shown below for each group following reserpine essentially provide the same information, don't concern yourselves with any differences.

c. Compare the effect of α-h-CRF on TH levels following exposure to environmental stressors vs. exposure to reserpine. What does this information suggest about regulation of TH gene expression?

The CRF antagonist blocked the impact of a stressor on TH levels in the LC but had no effect on the induction of TH by exposure to the drug reserpine. This indicates that there are multiple signaling mechanisms via which TH expression can be regulated.

9. (10 pts.) Progesterone has been shown to be an antianxiety agent. The elevated plus-maze is often utilized as a test of anxiety in rats. An antianxiety agent is one that increases the amount of time rats spend on the open arms of the maze. The ability of progesterone to enhance open arm performance is illustrated In fig. 1 (black bars indicate acute exposure to progesterone and open bars indicate acute exposure to vehicle). Prior to progesterone or vehicle, one group of animals had been pretreated with the GABA_A receptor antagonist, picrotoxin, while another group had been pretreated with the progesterone receptor antagonist, RU-38486. In fig. 2, the effect of progesterone (black bars) on plus-maze performance was evaluated in animals pre-exposed to either vehicle or 4-MA, an inhibitor of the enzyme 5-alpha reductase.

a. Does this data implicate a genomic or non-genomic effect of progesterone in mediating antianxiety? Explain your reasoning and indicate the critical data that support your conclusion. What is the critical mechanism essential for this action of progesterone?

The data indicate that progesterone mediates antianxiety effects via a non-genomic mechanism. The first set of data showed that pretreatment with the progesterone receptor antagonist, RU-38486, did not prevent the antianxiety effect of progesterone. Now, hormone receptors are cytoplasmic receptors and when bound with hormone they become transcription factors, regulating gene expression. The lack of effect of blocking the progesterone receptor on the antianxiety effect of progesterone then ruled out a direct genomic effect of the hormone. The first set of data also showed that blocking function of the GABA_A receptor with picrotoxin did prevent the anxiolytic effect of progesterone. This suggests that progesterone elicits its antianxiety effect via affecting function at GABA_A receptors, membrane receptors. Furthermore, the data in Fig. 2 indicated that it is not progesterone itself that is acting at GABA_A receptors, but rather it is reduced metabolites of progesterone that act at the receptors. This is indicated by the observation that blocking the enzyme that reduces progesterone to metabolites that can act at GABA_A receptors prevented the anxiolytic effect of progesterone. Since the anxiolytic effect of progesterone then appears to relate to modification of function of a membrane receptor and not to a direct genomic action of the hormone, a non-genomic effect is implicated.