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Why the Thrill is Gone: Scientists Identify Potential Target for Treating Major Symptom of Depression

December 16, 2012 1 comment

Stanford University School of Medicine scientists have laid bare a novel molecular mechanism responsible for the most important symptom of major depression: anhedonia, the loss of the ability to experience pleasure. While their study was conducted in mice, the brain circuit involved in this newly elucidated pathway is largely identical between rodents and humans, upping the odds that the findings point toward new therapies for depression and other disorders.

Additionally, opinion leaders hailed the study’s inventive methodology, saying it may offer a much sounder approach to testing new antidepressants than the methods now routinely used by drug developers.

While as many as one in six Americans is likely to suffer a major depression in their lifetimes, current medications either are inadequate or eventually stop working in as many as 50 percent of those for whom they’re prescribed.

“This may be because all current medications for depression work via the same mechanisms,” said Robert Malenka, MD, PhD, the Nancy Friend Pritzker Professor in Psychiatry and Behavioral Sciences. “They increase levels of one or another of two small molecules that some nerve cells in the brain use to signal one another. To get better treatments, there’s a great need to understand in greater detail the brain biology that underlies depression’s symptoms.” The study’s first author is Byung Kook Lim, PhD, a postdoctoral scholar in Malenka’s laboratory.

Malenka is senior author of the new study, published July 12 in Nature, which reveals a novel drug target by showing how a hormone known to affect appetite turns off the brain’s ability to experience pleasure when an animal is stressed. This hormone, melanocortin, signals to an ancient and almost universal apparatus deep in the brain called the reward circuit, which has evolved to guide animals toward resources, behaviors and environments — such as food, sex and warmth — that enhance their prospects for survival.

Scientists found that both chronic stress and the direct administration of melanocortin diminished the signaling strength of some synapses in the nucleus accumbens that contain receptors for melanocortin. The nucleus accumbens is labeled in this drawing of a human brain cross section. (up)

“This is the first study to suggest that we should look at the role of melanocortin in depression-related syndromes,” said Eric Nestler, MD, PhD, professor and chair of neuroscience and director of the Friedman Brain Institute at Mount Sinai School of Medicine in New York. Nestler was not involved in the study but is familiar with its contents. Read more…

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Common Parasite May Trigger Suicide Attempts

September 5, 2012 Leave a comment

A parasite thought to be harmless and found in many people may actually be causing subtle changes in the brain, leading to suicide attempts.

New research appearing in the August issue of The Journal of Clinical Psychiatry adds to the growing work linking an infection caused by the Toxoplasma gondii parasite to suicide attempts. Michigan State University’s Lena Brundin was one of the lead researchers on the team.

About 10-20 percent of people in the United States have Toxoplasma gondii, or T. gondii, in their bodies, but in most it was thought to lie dormant, said Brundin, an associate professor of experimental psychiatry in MSU’s College of Human Medicine. In fact, it appears the parasite can cause inflammation over time, which produces harmful metabolites that can damage brain cells.

“Previous research has found signs of inflammation in the brains of suicide victims and people battling depression, and there also are previous reports linking Toxoplasma gondii to suicide attempts,” she said. “In our study we found that if you are positive for the parasite, you are seven times more likely to attempt suicide.”

The work by Brundin and colleagues is the first to measure scores on a suicide assessment scale from people infected with the parasite, some of whom had attempted suicide.

The Toxoplasma gondii parasite has been linked to inflammation in the brain, damaging cells. Image adapted from MSU press release image. (up)

The results found those infected with T. gondii scored significantly higher on the scale, indicative of a more severe disease and greater risk for future suicide attempts. However, Brundin stresses the majority of those infected with the parasite will not attempt suicide: “Some individuals may for some reason be more susceptible to develop symptoms,” she said.

“Suicide is major health problem,” said Brundin, noting the 36,909 deaths in 2009 in America, or one every 14 minutes. “It is estimated 90 percent of people who attempt suicide have a diagnosed psychiatric disorder. If we could identify those people infected with this parasite, it could help us predict who is at a higher risk.”

T. gondii is a parasite found in cells that reproduces in its primary host, any member of the cat family. It is transmitted to humans primarily through ingesting water and food contaminated with the eggs of the parasite, or, since the parasite can be present in other mammals as well, through consuming undercooked raw meat or food.

Brundin has been looking at the link between depression and inflammation in the brain for a decade, beginning with work she did on Parkinson’s disease. Typically, a class of antidepressants called selective serotonin re-uptake inhibitors, or SSRIs, have been the preferred treatment for depression. SSRIs are believed to increase the level of a neurotransmitter called serotonin but are effective in only about half of depressed patients.

Brundin’s research indicates a reduction in the brain’s serotonin might be a symptom rather than the root cause of depression. Inflammation, possibly from an infection or a parasite, likely causes changes in the brain’s chemistry, leading to depression and, in some cases, thoughts of suicide, she said.

“I think it’s very positive that we are finding biological changes in suicidal patients,” she said. “It means we can develop new treatments to prevent suicides, and patients can feel hope that maybe we can help them.

“It’s a great opportunity to develop new treatments tailored at specific biological mechanisms.”

References:

Source: Michigan State University press release
Image Source: T. gondii image adapted from Michigan State University press release image
Original Research: Abstract and full paper from MSU (PDF file) for “Toxoplasma gondii Immunoglobulin G Antibodies and Nonfatal Suicidal Self-Directed Violence” by Yuanfen Zhang, MD, PhD; Lil Träskman-Bendz, MD, PhD; Shorena Janelidze, PhD; Patricia Langenberg, PhD; Ahmed Saleh, PhD; Niel Constantine, PhD; Olaoluwa Okusaga, MD; Cecilie Bay-Richter, PhD; Lena Brundin, MD, PhD; and Teodor T. Postolache, MD in Journal of Clinical Psychiatry online July 2012 73(8):1069–1076 doi: 10.4088/JCP.11m07532

Dorsoventral vs. Septotemporal hippocampus


Everybody knows what the hippocampus is for: memory. And…maybe something about anxiety or depression? Yes – over the last 10 years or so many studies have been published showing that the hippocampus has these two roles and that the mnemonic and emotional functions of the hippocampus are associated with its septal (dorsal) and temporal (ventral) ends, respectively. This new knowledge means that we’ve had to reorient our perspective. What we see when we consider the septal hippocampus may not be the same if we only consider its temporal end. My goal here is not to provide a review of the memory vs. emotional functions of the hippocampus (btw this dichotomy is a vast oversimplification). Instead, I’d like to talk about how people have differentiated these two ends of the hippocampus in their analyses. I’m also happy to showcase a bunch of pretty anatomical images that will probably never be published in a traditional journal article.

Some studies showing different functions of septal and temporal hippocampus

  • Some of the best reviews of the topic are by Bannerman et al from 2004 and 2011.
  • A recent and free review article by Fanselow and Dong.
  • Classic Moser papers showing spatial memory is more dependent on dorsal hippocampus and anxiety/fear behavior on ventral hippocampus
  • recent paper suggesting that spatial processing in the septal hippocampus meets the behavioral-control functions of the temporal hippocampus to enable rapid spatial learning

History of neurogenesis quantification. So, back in the day, before I even knew what a neuron was, and before it was well-established that there was functional differentiation along the hippocampal axis, people would pick a few sections from the dorsal hippocampus (it’s much more photogenic, gets all the glory), count new neurons, and make it a density measurement. Then the stereology police arrived (seriously, that’s what they’re called) and pointed out that changes in tissue volume or cell packing could change density measurements without there being any differences in numbers of cells. Stereological analyses also prevent any biases that might arise from creating arbitrary boundaries when examining only part of the hippocampus. And so people started doing stereological counts, which require a systematic quantification throughout the entire hippocampus. My guess is that this probably delayed the appreciation that neurogenesis could vary in magnitude and function along the hippocampal axis. Now that we know that stereology is pointless we can get back to business (this is a joke – please don’t arrest me).

Difficulty of quantifying subregions due to curvature of the hippocampus. One of the reasons the hippocampus is such a popular neurobiological model is its anatomy – the dentate gyrus, CA3 and CA1 subfields are all composed of tightly packed cells that are easy to identify. Thinking of the hippocampus along its long axis, one end projects to the septum and the other abuts the temporal lobe, hence “septotemporal” is technically the most accurate way to refer to the different ends of the hippocamus. The hippocampus is curved in such a way that you can actually cut it along any of the 3 spatial planes (X, Y, Z aka coronal, horizontal, saggital) and hit the hippocampus perpendicular to the septotemporal axis somewhere, giving rise to the classic the trisynaptic circuit. However, because of this same curvature, sectioning the brain in only one of the three planes means that some portion of the hippocampus is not going to be cut perpendicular to the long axis, producing sections in which septotemporal coordinates are hard to define.

The 3D nature of the hippocampus using images from the Allen Brain Explorer:

Figure 1: The dentate gyrus subfield of the hippocampus (i.e. green banana), from its septal pole, extends caudally and laterally and then ventrally. Green axis=dorsoventral, red=rostrocaudal, yellow=mediolateral.

Figure 2: A relatively caudal coronal section with the 3D dentate gyrus shown in the left panel, for comparison. This section contains ventral dentate gyrus (at the bottom, by “temporal”) but, at the top of the section, it also contains a portion of the dentate gyrus that is very dorsal, despite being far from the septal pole.

Figure 3: This section is more caudal than the previous example, yet the dentate granule cells (white patches within the bright green region) do not extend as far in the ventral direction. So, more caudal ≠ more ventral.

Others on the curvature problem:

Schlessinger et al., 1975: Since the dentate gyrus follows the general curvature of the hippocampal formation, it is difficult to apply the usual topographical terms to its various parts. The rostral third or half of the gyrus is more-or-less horizontally disposed within the cerebral hemisphere…At about the junction of its rostral and caudal halves the gyrus is sharply flexed upon itself, and comes to be vertically disposed….Again, because of the flexure of the hippocampal formation, it is inappropriate to refer to the dentate gyrus as having a dorsal (or rostral) and a ventral (or caudal) part. Following Gottlieb and Cowan (’73) we shall refer to the long axis of the gyrus, extending from the temporal pole of the hemisphere to just behind the septal region, as its temporalseptal axis.

Amaral & Witter, 1989: Because of its complex three-dimensional shape, normal sections of the hippocampus, i.e. those oriented perpendicular to the long axis, are obtained for only a small part of its septotemporal extent in standard coronal or horizontal sections. This situation severely complicates the analysis of the connections within the hippocampal formation.

De Hoz et al., 2003: In discussing different regions of the hippocampus, we use the terms “septal” and “temporal” to refer to the rostralmost and the ventralmost poles of the longitudinal axis, respectively, because this terminology allows an even division of this axis into septal and temporal halves. The terms “dorsal” and “ventral” are sometimes used to refer to the same areas; the dorsal hippocampus is, however, more extensive than the ventral.

So how can we divide the hippocampus? Many people work with coronal sections. Can we delineate boundaries between different hippocampal subregions in coronal sections? Banasr et al. has described a reproducible method for separating dorsal from ventral hippocampus using coronal sections. Here, the dorsal regions would contain a fair bit of mid-septotemporal hippocampus but indeed, only the dorsal sections would contain septal hippocampus and only ventral sections would contain temporal hippocampus:

Figure 4: Separating dorsal and ventral hippocampus in coronal sections

Jayatissa et al. has horizontally sectioned the rat brain and then used anatomical coordinates to divide dorsal from ventral. This seems to be a good way to isolate pure, septal hippocampus but dorsal measures would again blur together the septal and mid-septal regions.

What if we wanted to separate the septal and temporal ends of the hippocampus? One method, described in Amaral & Witter, 1989 offers a solution:

We have adopted a strategy first described by Gaarskjaer that obviates this problem. In short…the fixed hippocampal formation is dissected from the brain and gently extended before histological processing. In this way the extended hippocampus can be positioned such that normal sections are obtained from much of the septotemporal extent of the structure.

A similar approach had been used (see here and here). One drawback is that you ruin much of the rest of the brain during the dissection process (insert but-who-cares-about-the-rest-of-the-brain joke here). Here’s a figure from thesis that illustrates the similar-shaped hippocampal slices obtained with this method:

Figure 5: DAPI counterstained sections, evenly spaced across the septotemporal axis. Sampling scheme illustrated at the top. Shaded regions indicate how different septotemporal regions could be binned. S=suprapyramidal blade of the dentate gyrus, I=infrapyramidal blade, DG=dentate gyrus.

Another strategy isn’t too different from the method of Banasr, above. To get at the septal hippocampus you’re just being a bit more selective and only examining portions of the dorsal hippocampus that extend quite far rostrally. For the caudal sections that contain both dorsal and ventral hippocampus the rhinal fissure seems like a good guide – anything falling on the ventral side I’m counting as ventral.

But if you’re lazy…

A fast, revolutionary new method for examining the hippocampus along its full septotemporal axis in a single section! It almost sounds too good to be true. In fact, it is. But it provides some interesting pictures for those of you who have stuck with me this far.

Recently, a lot of rats has been irradiated to eliminate adult neurogenesis. Before coming to any conclusions about the behavioral data it was needed to know whether neurogenesis was completely blocked AND whether it was blocked throughout the entire dentate gyrus. Due to laziness to cut hundreds of sections for each rat, the hippocampus was extracted but instead of sectioning perpendicular to its septotemporal axis, it was sectioned parallel to, or along, its septotemporal axis by flattening and freezing it on a microtome stage. With this approach the entire dentate gyrus could be cut in about 30 sections and sections that had the entire septotemporal length of the dentate gyrus became present. Then they were stained for NeuN and DCX to visualize neurons and immature neurons, respectively. I think every other section was stained; one example is shown below.

Figure 6: Hippocampal sections stained for NeuN and DCX. The dentate gyrus can be identified as the layer of tightly-packed orange cells on the left, that are bordered by green DCX+ cells. Sections were cut from the side of the infrapyramidal blade towards the suprapyramidal blade (direction of cutting = section 1→9). Images were taken with a 20x objective and subsequently stitched together.

Is it really necessary to divide septotemporally? I guess it depends. Many studies that have focussed more on dorsal vs. ventral have made significant findings. If the anatomical method is well-described and reproducible, what more could you ask for? It’s possible, however, that combining different septotemporal regions into the same analysis could obscure a result. For example, when the activation of new neurons was examined after water maze training, it was found a steadily-increasing amount of activation as going from septal to temporal (see Figure 7). Had the 2 septal quartiles been pooled together and the 2 temporal quartiles pooled together, the observed difference would have been much smaller than when comparing the septalmost quartile with the temporalmost quartile.

Figure 7: The density of ‘activated’ new neurons (i.e. PSA-NCAM+ and Fos+) increased from septal to temporal. Note the mid-septal and mid-temporal regions were similar. Also note that I used D and V nomenclature, for ‘dorsal’ and ‘ventral’, despite repeatedly emphasizing in this post that ’septal’ and ‘temporal’ is more accurate.

——————————————————————————-

and now…

Pretty pictures from these sections!

Messy.

Just a nice example of some DCX dendrites.

DCX labeling outside of the dentate gyrus. I think this was in the subiculum but who can say for sure with these weird sections.

Septotemporal sample #1

Septotemporal sample #2

Septotemporal sample #3

Septotemporal sample #4

CRAB

ALLIGATOR

PUPPY / BIRDIE

Special thanks and credit to http://www.functionalneurogenesis.com and to Sarah Ferrante for sectioning, staining and imaging the tissue.

Ketamine for Depression: Yay or Neigh?

December 14, 2010 Leave a comment

Venn diagram of psychoactive drugs (up)

 

NOTE: This post is part of a Nature Blog Focus on hallucinogenic drugs in medicine and mental health, inspired by a recent Nature Reviews Neuroscience paper, The neurobiology of psychedelic drugs: implications for the treatment of mood disorders, by Franz Vollenweider & Michael Kometer. This article will be freely available, with registration, until September 23. For more information on this Blog Focus, see the Table of Contents.

 

Veterinary Anesthetic, Club Drug, or Antidepressant?
Club drug “Special K” (aka ketamine) is stepping out of the laser light into the broad daylight of mainstream psychiatry with the publication of a new review article by Vollenweider and Kometer (2010). Long used to anesthetize animals (and children), ketamine was classified as a “dissociative anesthetic” by Domino et al. (1965) for its combined effects of sedation/analgesia and hallucinations. Domino (2010) recently revisited his classic paper, which reported on a study in 20 volunteers incarcerated at the Jackson Prison in Michigan:

The first human was given ketamine in an intravenous subanesthetic dose on August 3, 1964. Guenter [Corssen, M.D.] and I gradually increased the dose from no effect, to conscious but “spaced out,” and finally to enough for general anesthesia. Our findings were remarkable! The overall incidence of side effects was about one out of three volunteers. Frank emergence delirium was minimal. Most of our subjects described strange experiences like a feeling of floating in outer space and having no feeling in their arms or legs.

The ego death of the “K hole” can be a terrifying experience for some (“I ceased to exist”) or transformative for others (“I witnessed myself as a part of the universal collective of strange energy”)1. In their Nature Reviews Neuroscience opinion piece, Vollenweider and Kometer considered ketamine a psychedelic, along with the traditional hallucinogens such as LSD, psilocybin, and mescaline. They noted that both classes of drugs may have psychotherapeutic effects through actions on the excitatory glutamate neurotransmitter system.

Ketamine is an antagonist of the glutamate NMDA receptor and is thought to work by blocking NMDA receptors on inhibitory GABA-containing interneurons, ultimately promoting glutamate release. In a scientific tour de force, Li and colleagues (2010) demonstrated that the mTOR (mammalian target of rapamycin) protein kinase pathway is rapidly activated by ketamine. This sets off a cascade of events including the formation of new synapses on dendritic spines. Using a combination of cellular, molecular, electrophysiological, behavioral, and phamacological techniques, ketamine was shown to exhibit antidepressant properties in animal models of depression and anxiety, perhaps via rapid induction of synaptic plasticity in the medial prefrontal cortex (PFC). Regions of the medial PFC in humans, particularly the ventral anterior cingulate cortex, have been implicated in the pathophysiology of major depression.

Human clinical trials of ketamine as a rapidly acting antidepressant aren’t especially new. A randomized, double-blind study in 2000 involved administration of saline or a single subanesthetic dose of ketamine (0.5 mg/kg intraveneously) to nine depressed patients, seven of whom completed the trial (Berman et al., 2000). Within 72 hrs, amelioration of depressive symptoms was observed. Half of the treated patients showed a 50% or greater improvement in depression scores. However, these therapeutic effects weren’t very long-lasting, returning to baseline levels in 1-2 weeks. In a larger study, 18 patients with major depression participated in a similar double-blind cross-over design where they received the 0.5 mg/kg dose of ketamine and placebo one week apart (Zarate et al., 2006). The patients were rated at baseline and at 40, 80, 110, and 230 minutes and 1, 2, 3, and 7 days post-infusion on a number of clinical scales, including the Hamilton Depression Rating Scale (HDRS), the Brief Psychiatric Rating Scale (BPRS) positive symptoms subscale, and the Young Mania Rating Scale (YMRS).

The primary outcome measure was change in HDRS score, shown in Figure 2 below (top graph). Significant improvements began at the 110 min time point. Scores declined further from 1-3 days and remained below placebo levels for 7 days. However, unusual experiences were noted at 40 min, with substantial increases in scores for psychosis-like and mania-like symptoms. Other adverse events associated with ketamine included…

…perceptual disturbances, confusion, elevations in blood pressure, euphoria, dizziness, and increased libido. … The majority of these adverse effects ceased within 80 minutes after the infusion. In no case did euphoria [YMRS] or derealization/depersonalization [BPRS] persist beyond 110 minutes (Figure 2, middle and bottom graphs).


Figure 2 (Zarate et al., 2006). Change in the 21-item HDRS, BPRS positive symptoms subscale, and YMRS scores over 1 week (n=18). Values are expressed as generalized least squares means and standard errors for the completer analysis. * indicates P<.05; †, P<.01; ‡, P<.001.

 

So here we have several research groups that say yay! to ketamine as an antidepressant. Are there any naysayers?

Although the immediate onset of symptom amelioration gives ketamine a substantial advantage over traditional antidepressants (which take 4-6 weeks to work), there are definite limitations (Tsai, 2007). Drawbacks include the possibility of ketamine-induced psychosis (Javitt, 2010), limited duration of effectiveness (aan het Rot et al., 2010), potential long-term deleterious effects such as white matter abnormalities (Liao et al., 2010), and an inability to truly blind the ketamine condition due to obvious dissociative effects in many participants.

At present, what are the most promising uses for ketamine as a fast-acting antidepressant? Given the disadvantages discussed above, short-term use for immediate relief of life-threatening or end-of-life depressive symptoms seem to be the best indications.

Suicidal Ideation
Acute ketamine treatment in suicidal patients presenting at the ER has the potential to provide immediate changes in the risk that a patient will harm herself when released, when accompanied by proper followup and appropriate long-term treatment. An open label study in 33 patients with refractory depression involved infusion of 0.5 mg/kg ketamine over a period of 40 min (DiazGranados et al., 2010). Those with high scores on the Scale for Suicide Ideation showed significant improvements at 40 min that were maintained for the 230 min duration of the study. Obviously, one would like to follow actively suicidal patients for a longer period of time than 4 hrs, and future clinical trials should take this into account.

Palliative Care
Watching a terminally ill loved one suffer from unbearably excruciating pain is one of the most emotionally wrenching experiences you’ll ever have. Anything, and I mean anything , that will relieve this sort of suffering should be freely administered without reservation or stigma. As discussed in The secret history of psychedelic psychiatry, psilocybin has been shown to alleviate anxiety and pain in cancer patients. Reports of psychedelic psychotherapy in the 60s and 70s suggested that many patients overcame their fear of death through LSD-facilitated sessions. More recently, an open label study in two hospice patients, each with a prognosis of only weeks or months to live, showed beneficial effects of ketamine in the treatment of anxiety and depression (Irwin & Iglewicz, 2010). A single oral dose produced rapid improvement of symptoms and improved end of life quality. To disentangle the pain relieving and antidepressant effects of ketamine, the authors emphasized the importance of conducting clinical trials for this particular indication.

Better Drugs for a Brighter Tomorrow
Newer NMDA antagonist drugs with fewer dissociative side effects (e.g., more selective antagonists such as NR2B receptor blocker EVT 101) are undergoing testing and development. Personalized medicine and pharmacogenomics may ultimately shift psychedelic experiences out of the realm of hippies and into the doctor’s arsenal.

 

References

aan het Rot M, Collins KA, Murrough JW, Perez AM, Reich DL, Charney DS, Mathew SJ. (2010). Safety and efficacy of repeated-dose intravenous ketamine for treatment-resistant depression. Biol Psychiatry 67:139-45.

Berman RM, Cappiello A, Anand A, Oren DA, Heninger GR, Charney DS, Krystal JH. (2000). Antidepressant effects of ketamine in depressed patients. Biol Psychiatry 47:351-4.

DiazGranados N, Ibrahim LA, Brutsche NE, Ameli R, Henter ID, Luckenbaugh DA, Machado-Vieira R, Zarate CA Jr. (2010). Rapid resolution of suicidal ideation after a single infusion of an N-methyl-D-aspartate antagonist in patients with treatment-resistant major depressive disorder. J Clin Psychiatry. Jul 13. [Epub ahead of print]

Domino EF. (2010). Taming the ketamine tiger. Anesthesiology 113:678-84.

Domino EF, Chodoff P, Corssen G. (1965). Pharmacologic Effects of CI-581, a New Dissociative Anesthetic, in Man. Clin Pharmacol Ther. 6:279-91.

Irwin SA, Iglewicz A. (2010). Oral ketamine for the rapid treatment of depression and anxiety in patients receiving hospice care. J Palliat Med. 13:903-8.

Javitt DC. (2010). Glutamatergic theories of schizophrenia. Isr J Psychiatry Relat Sci. 47:4-16.

Li N, Lee B, Liu RJ, Banasr M, Dwyer JM, Iwata M, Li XY, Aghajanian G, Duman RS. (2010). mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists. Science 329(5994):959-64.

Liao Y, Tang J, Ma M, Wu Z, Yang M, Wang X, Liu T, Chen X, Fletcher PC, Hao W. (2010). Frontal white matter abnormalities following chronic ketamine use: a diffusion tensor imaging study. Brain 133:2115-22.

Tsai GE. (2007). Searching for rational anti N-methyl-D-aspartate treatment for depression. Arch Gen Psychiatry 64:1099-100; author reply 1100-1.

Vollenweider, F., & Kometer, M. (2010). The neurobiology of psychedelic drugs: implications for the treatment of mood disorders Nature Reviews Neuroscience, 11 (9), 642-651 DOI: 10.1038/nrn2884

Zarate CA Jr, Singh JB, Carlson PJ, Brutsche NE, Ameli R, Luckenbaugh DA, Charney DS, Manji HK. (2006). A randomized trial of an N-methyl-D-aspartate antagonist in treatment-resistant major depression. Arch Gen Psychiatry 63:856-64.