Histology staining of three dopamine-synthesizing sources in the hypothalamus support the findings in Figure 5E. The amount of TH cells (dopamine-synthesizing cells) in these regions decreases with 6-OHDA and can be partially rescued with short-day photoperiod exposure.

Even with 6'OHDA treatment, exposing the animals to short-day photoperiod resulted in some behavioral rescue, reflected by the animals spending more time spent in the open arm in the EPM test and less time spent immobilized in the forced swim test. The difference in behavior is significant comparing results of the short- vs long-day photoperiod exposure. One additional comparison that could be insightful are the results of 6'OHDA on 12L:12D photoperiod exposure; these can be found in Figure 5 B, and do not appear to be significantly different from the long-photoperiod exposure results. Thus, a follow up idea for the researchers is to consider when the effect of increasing the duration of light-exposure in a day begins to saturate.

Here, dopamine synthesis is being stopped by two things: the neurotoxic agent 6-OHDA and long-day light exposure, a stressor for nocturnal animals. Both treatments should reduce the presence of dopamine-making neurons.

The periventricular nucleus is a thin sheet of neurons located in the hypothalamus. For the purpose of this paper, PeVN can be defined as the part of the hypothalamus that is a major source of dopamine and somatostatin expression.

The paraventricular nucleus is a region of the hypothalamus, considered to be the body's most important autonomic control center. The neurons of the PaVN are involved in controlling stress, metabolism, growth, reproduction, immune, gastrointestinal, renal, and cardiovascular functions. For the purpose of this paper, PaVN can be defined as the part of the hypothalamus that is a major source of dopamine and somatostatin expression.

placebo

These findings validate the specificity of 6-OHDA's delivery. In the dopaminergic cell group A13 just 200 μm away from the treatment site, the effects of 6-OHDA are not observed.

The dopamine receptor antagonist sulpiride functions by binding to the D2 dopamine receptor to block dopamine from binding.

SCH23390 functions by binding to the D1 dopamine receptor to block dopamine from binding.

These findings validate the effectiveness of TH-IR ablation by 6-OHDA, as a decrease in time spent in the open arm of the EPM is associated with increased anxiety, and an increase in immobility time in the FST is associated with a depressed state. These results are not surprising as it is expected that a halt in the synthesis of dopamine (the feel-good transmitter) would increase anxiety and depression.

Following action potential firing, transmitters will be sent from the presynaptic, or sending, neuron to the postsynaptic, or receiving, neuron; the received transmitter will thereby alter the likelihood of that postsynaptic neuron firing its own action potential. The experimenters had observed that at certain clusters of neurons in the CNS, short photoperiod exposure increased production of the transmitter dopamine, and long photoperiod exposure led to an increased production of somatostatin. Consequently, the experimenters are questioning whether expression of those cognate postsynaptic receptors, i.e., dopamine receptors or somatostatin receptors, follow suit.

a neurotoxic synthetic organic compound used by researchers to selectively destroy the brain's dopaminergic as well as noradrenergic neurons. In this paper, it was used to ablate dopaminergic neurons, so as to stop dopamine synthesis.

a control treatment that is similar to the experimental treatment, but omits the key therapeutic element being tested.

For rats, nocturnal mammals, increased photoperiod exposure is a stressor. Consequently, in both the EPM and FST, rats exhibit more depressive/anxious behaviors following long photoperiod exposure. In the EPM test, these rats spend less time in the open arm, and in the FST, they give up swimming and become almost immobile much sooner than the control group. For the short-day exposure group, the rats have more open arm activity, and in the FST persevere for longer, indicating reduced anxiety relative to the control group.

In 1977, Porsolt et al. described a new method for assessing the effectiveness of antidepressent treatments, in which mice are dropped into a cylinder of water with a diameter of 10 cm, height of 25 cm, and water height of 6 cm. Porsolt et al. had observed that most mice are able to find the exit within 10 minutes, but some display a "state of despair", where they believe there is no escape from the situation, and resort to simply floating, only making movements that are needed to keep their head above the water. Porsolt et al. used this method to test a large range of antidepressants by injecting the mice intraperitoneally 1 hour before the forced swim test. In Porsolt et al.'s results, three drugs known to be therapeutic in depression (iprindole, mianserin, and viloxazine) resulted in a reduction in immobility of the mice (32).

The elevated plus maze has been widely used to assay the anti-anxiety effects of certain drugs on rodents. The EPM is comprised of four arms that are oriented in an "X"; two of the arms are open/without walls and two are enclosed by walls. The rat would be placed at the junction of those four arms, with a starting position that has them face an open arm. During the course of five minutes, a video-tracking system as well as the observer will record the number of entries and duration the rodent spends in each arm. An increase in open arm activity (duration or entries) is viewed as anti-anxiety behavior (31).

Panel D of Figure 4 shows that CSF levels of CRF and plasma levels of corticosterone significantly decrease following short photoperiod exposure. In panel B, short photoperiod exposure results in elevated coexpression of D2R and SST2/4R. These findings support the hypothesis that increased coexpression of D2R and SST2/4R has inhibitory effects on CRF neurons of the third ventricle.

When expression of a receptor is greater than that of its transmitter, the likelihood of those transmitters binding to the receptor will be high. Thus, if levels of SST2/4R are high compared to the presynaptic SST, then presynaptic SST will likely be able to bind to the cognate receptor, and induce its inhibitory effects.

Since D2R and SST2/4R are linked to inhibitory effects, there increased coexpression in CRF neurons might inhibit CRF production.

The experimenters used fluorescent antibodies specific for binding to CRF to ensure that they are examining CRF neurons, as previous work indicates that SST and dopaminergic interneurons send their transmitters to CRF neurons. The experimenters also perform staining with fluorescent antibodies specific for SST2/4R, the somatostatin receptor, and D2R, the dopamine receptor.

The experimenters' measurements of mRNA expression are used to determine whether transmitter respecification is resulting from new transcription or preexisting transcription. If transmitter respecification is resulting from preexisting transcripts, TH and SST mRNA would already be present in the cytosol and only following exposure to a certain photoperiod condition, translational machinery would translate that existing mRNA. Thus, if the source is preexisting mRNA, then we should not observe TH and SST mRNA counts follow numbers of TH and SST neurons, and instead mRNA levels should be unchanged. The fact that the experimenters are observing that mRNA levels follow TH and SST neuron count demonstrates that the photoperiod exposure condition is inducing de novo transcription, rather than direct translation of preexisting transcripts.

Previous work establishes that somatostatin signaling is activated by different stressors, and describes somatostatin as having anti-stress effects by blunting endocrine, autonomic, behavioral, and visceral gastrointestinal motor responses (24). Nocturnal animals function optimally in dark conditions; thus, an increase in day exposure would be considered a stressor for them. Somatostatin is, therefore, expected to be elevated in the long day exposure condition.

this technique enables measurement of gene expression in single cells by detecting and counting individual RNA molecules.

Because one role of VMAT2 is to package dopamine from the cytosol into synaptic vesicles for their release from the neuron, VMAT2 serves as a marker for the presence of dopamine. In addition to VMAT2 expression, the expression of the dopamine transporter, DAT, was also observed. Altogether, these findings serve as evidence that the newly expressed TH-IR neurons, induced by short-day photoperiod exposure, are indeed synthesizing dopamine.

Panel D is consistent with the findings in Panel B and Panel C, which also demonstrate an increase in SST expression following increase day exposure.

places along the length of an axon, other than the axon terminal, that join with other neurons.

In response to the photoperiod exposure condition, the circuit activity changes such that the neurons of the reserve pool are switching transmitters. The researchers believe that the TH-IR and SST-IR neurons are coming from this reserve pool of cells based on their finding that short-day exposure led to an increase in the recruitment of TH-IR neurons and diminishment of SST-IR neurons, while long-day exposure had the opposite effect.

In a review article on reserve pool neuron transmitter respecification, Dulcis et al. provide a helpful analogy for understanding reserve pools. In this analogy, the role of reserve pools is compared to having two jobs, and following certain physiological stimuli, one of those jobs is relinquished. Dulcis et al. defines reserve pool neurons as "cells that share inputs and outputs with adjacent core pools of neurons but express different neurotransmitters." In one situation, the neurons from both pools could be expressing the same transmitters, but the core neurons also express a secondary transmitter; following the change in circuit activity, the neurons of the reserve pool will stop expressing the transmitter that it has in common with the core pool of neurons. In an alternative scenario, these two pools of neurons could be expressing different neurotransmitters, and the change in circuit activity results in the neurons of the reserve pool acquiring the expression of the transmitter that is already expressed by the core neurons (23).

The experimenters performed immunostaining for both dopamine and SST expression. Panel B depicts immunofluorescence of these transmitters in the PaVN. The first of the three images depicts the results from long day exposure; under this condition, there is an abundance of red representing an increase of SST expression and very little green indicating that there is a decrease in dopamine expression. Under short day exposure (third image), the opposite is true.

Finding that the TH-IR neuron counts are reversible upon application of opposite photoperiod treatment is further evidence that sensory stimuli can induce neuroplastic changes in the already mature brain.

During the late stages of apoptosis, cells undergo extensive DNA degradation. The resulting 3′- hydroxyl ends of these double-strand DNA breaks can be attached with nucleotides by the enzyme terminal deoxynucleotidyl transferase. In the TUNEL staining employed by the experimenters, the nucleotides attached by TdT are tagged directly with a fluorescent label. All in all, the TUNEL assay serves as a measurement for apoptosis.

BrdU labeling did not result in significant amounts of active poliferating cells; this is an initial indication that inverse SST and dopamine expression is not due to neurogenesis.

active during the day; opposite of nocturnal.

BrdU is an analog of the nucleoside thymidine, and would be incorporated into newly synthesized DNA. Since the fundamental requirement for cell proliferation is DNA synthesis, quantifying the incorporation of BrdU following application of anti-BrdU antibodies will therefore enable measurements of cell proliferation.

A study elucidating the counter-regulatory mechanisms used to maintain energy balance in response to environmental or physiological stressors revealed that neurons important for energy homeostasis can be regenerated in adult feeding centers (a region of the hypothalamus). The results of Kokoeva et al. indicate that de novo neurogenesis might serve as a compensatory mechanism to regulate energy balance in the presence of environmental or physiological insults (22). Based on previous work, the researchers have decided to examine whether the inverse relationship they have found between somatostatin and dopamine numbers in the three photoperiods is due to neurons forming or from the neuron switching to produce more SST and less dopamine, or vice versa.

In order for the dopamine alterations to be induced by changes in light exposure duration, the dopaminergic neurons must be able to receive retinal input, as is the case with the dopaminergic neurons in the hypothalmic nuclei, innervated by the retino-hypothalmic projection. If the neurotransmitter respecificaiton is indeed due to the photoperiod treatment, dopamine production at an adjacent dopamine source, which does not receive retinal input via the SCN, should be unaffected.

The A13 group or A13 dopaminergic cell group consists of dopaminergic neurons residing in a region of the brain called the subthalamic nucleus.

our body's "internal clock" that regulates the sleep–wake cycle and repeats every 24 hours.

Within the three photoperiods, 19L:5D, 5L:19D, and 12L:12D, the number of TH-IR neurons are similar. Because there is a consensus within each photoperiod, this is indicating that circadian fluctuations are not contributing to neurotransmitter respecificaiton.

In panel C, before KCl depolarization, there is observable fluorescent signals from FFN511, indicating dopamine uptake. Upon KCl depolarization, the fluorescence is diminished; this is indicative of dopamine release. The results of panel C are consistent with panel A and B in that during the long day light condition (19 hours light, 5 hours darkness), the fluorescence signal is the lowest, which means less dopamine being taken up.

During synaptic vesicle fusion, the nervous system relies on neurotransmitters to transmit signals between neurons. Fluorescent false neurotransmitters have previously been used as substrates for VMAT2, and have served as probes for the imaging of dopamine release in the striatum (17).

A decreased amount of TH-IR neurons means that less dopamine is being produced; this was observed when the rats were given long-day exposure (19 hours of light, 5 hours of darkness). Short-day exposure (5 hours of light, 19 hours of darkness) resulted in the number of TH-IR neurons increasing, a sign of greater dopamine production. These results were all relative to the control group, rats that experienced a balanced-day with 12 hours of light and 12 hours of darkness.

VMAT2 is the CNS vesicular transporter that packages monoamines, such as dopamine, from the cytosol into synaptic vesicles for their release from the neuron.

Panel B: The fact that in all three photoperiod conditions, dopamine is colocalized with TH validates the use of TH as a marker of dopamine synthesis.

By staining brain tissue sections for an enzyme in the dopamine synthetic pathway, the experimenters can determine how much dopamine is being produced.

In 2009, Ashkenazy et al. performed experiments on Sand rats, diurnal animals, and showed that shortening their exposure to daylight resulted in anxious and depressed behaviors (11). In 2006, there was a study conducted at four Canada centers to compare the effectiveness of light therapy versus the antidepressant fluoxetine for patients with winter seasonal depression. The study suggested that exposure to placebo light 30 minutes/day had similar efficacy to the administration of fluoxetine (12). Since previous work had shown that for diurnal animals, shorter photoperiods can be anxiety or depression inducing (11), and longer light exposure can have anti-depression effects (12), the researchers have proceeded to hypothesize that longer photoperiods will have the opposite effect on nocturnal adult mammals.

anxious

Neuroplastic changes refers to the adjustments that neurons make in response to changes in their environment.

It is known that the young developing brain is able to add or switch the transmitters that their neurons express. The question guiding this research is whether sensory stimuli can cause the already mature (adult) brain to experience changes in the types of transmitters that are produced by their neurons.

The researchers found that neurotransmitters are not actually fixed upon maturation, but are, in actuality, dynamic and can be affected by sensory stimuli such as longer or shorter photoperiods. Changes in transmitter expression result in behavioral changes as well. Thus, follow up research can explore what other sensory stimuli can change transmitter expression and also examine the corresponding behaviors that are regulated by those transmitters.

Shortening the length of daylight the rats are exposed to induced the formation of new dopaminergic neurons and thereby restored the rats' previous (normal) behaviors, i.e., the rats no longer displayed anxious and depressed behaviors.

Neurons that produce the neurotransmitter dopamine. For mammals, this is the main source of dopamine in the CNS.

Using drugs to prevent dopamine production resulted in anxious and depressed behaviors by the rats; these same behaviors were also observed following extended day length exposure.

When more of the neurotransmitter dopamine is produced, there is also an increase in the postsynaptic dopamine receptor. Likewise, when less dopamine is produced, it follows that there is a decreased expression of dopamine receptors. However, for somatostatin, this was not the case. Regardless of whether somatostatin production is increased or decreased, the somatostatin receptor expression is unchanged.

exhibiting a phenotype (a set of features/behaviors) that does not correspond to one's own genotype, but rather is environmentally induced.

Altering the amount of time that a rat is exposed to light in a day leads to changes in the type of neurotransmitters being expressed by a set interneurons located in the hypothalamus. When the rat's daily light exposure is restricted to a shorter time frame, those interneurons favor dopamine expression. In contrast, longer daily periods of light exposure results in those same interneurons favoring somatostatin expression.

A photoperiod is the daily duration that an organism receives light exposure, i.e., length of day.

Interneurons connect sensory neurons, the nerve cells that convert environmental stimuli into internal electrical impulses, and motor neurons, which transmit signals from the brain to control muscle movements. Therefore, interneurons act as a "middle-man", passing signals from sensory neurons to motor neurons.

Commonly called the "feel-good" chemical because of its role in the brain's reward system, dopamine is a neurotransmitter associated with feelings of pleasure, learning, memory, and motor system functions.

Also known as growth hormone-inhibiting hormone, somatostatin is a peptide hormone that primarily functions to prevent the unnatural rapid proliferation of cells, the hallmark of tumors. Somatostatin also plays a role in the gastrointestinal system.

Neurotransmitters are signaling molecules used by the nervous system to transmit messages between neurons.