A new technology is making it possible to view the mammalian brain’s structure and connectivity for the first time. Karl Deisseroth discussed the technology, called CLARITY, at a plenary lecture at the 2014 meeting of the International College of Neuropsychopharmacology.
The way CLARITY works is by replacing lipids in the brain with a hydrogel substance. This preserves the structure of the brain’s neural networks, leaves proteins and nucleic acids intact, but allows for observation by rendering the brain transparent. This can be done in a system as large as the entire adult mouse brain. Early attempts took a whole day, but Deisseroth eventually found a way to render a mouse’s brain transparent in a matter of minutes.
The pictures are truly amazing, allowing for the visualization of previously microscropic neurons, dendrites, axons and connections in life-sized images. Pictures and details are available at www.clarityresourcecenter.org.
Deisseroth and colleagues have used CLARITY imaging to determine where neurons fire during different social activities. By placing photosensitive fibers in selected neurons using a virally based gene insertion technique, Deisseroth and colleagues were able to selectively fire dopamine neurons in the ventral tegmental area, part of the brain’s reward system, and thus increase or decrease the social interaction of mice by increasing or decreasing firing. The effects were selective to social interaction; the firing did not affect locomotor activity or exploration of an inanimate object.
The ventral tegmental area contains neurons that project to several locations in the brain, and Deisseroth and colleagues hoped to observe which were important to social interaction. Stimulating the ventral tegmental area to drive the medial prefrontal cortex caused anxiety in the mice and made them averse to social interaction. However, when the ventral tegmental area was used to selectively drive the nucleus accumbens, another part of the brain’s reward system, social interaction increased.
Deisseroth wanted to know if the nucleus accumbens was also involved in normal spontaneous social interactions. The researchers used a virus to insert an opsin-sensitive calcium gene that could give an ongoing readout of neural activity. (Opsin is a light-sensitive receptor found in cells in the retina.) The team found that the nucleus accumbens was implicated in social interaction with another mouse, but not in exploration of a novel object. Based on CLARITY imaging of the structure of ion channels (which are so small they cannot even be seen with an electron microscope), Deisseroth was able to selectively alter ion fluxes and turn neuronal firing on or off at will.
In the last 50 years, the brain and its billions of neurons and hundreds of trillions of synapses have gone from complete inaccessibility toward increasing clarity.
Researcher Tony Pitts presented a study at the 2014 meeting of the International College of Neuropsychopharmacology (CINP) that described the neurobiology of an animal model of depression in rodents. In animal models, researchers provoke depression-like symptoms in animals with the hopes of finding neurobiological clues to human depression. Pitts’ studies explored the effects of acute stressors as well as more chronic long-term stressors such as learned helplessness.
In the rodents, acute stressors caused increased cell firing in the hippocampus, which caused increases in burst firing and an increase in the number of cells firing in the ventral tegmental area, which then led to increased activity in the nucleus accumbens (the brain’s reward center). However, after the stressor was over, there was an opponent process that resulted in a much more prolonged period of inhibition in the nucleus accumbens, with associated decreases in psychomotor activity and reward seeking. The rodents lost their preference for sucrose and engaged in less intracranial self-stimulation, pressing a bar to stimulate the brain pleasurably. These and other effects suggest an analogy to anhedonia (loss of pleasure in activities that were previously enjoyed), which is a key component of human depression.
In related studies, after experiencing periods of inescapable shocks, rodents developed learned helplessness, failing to avoid the area where shocks were delivered even when an exit was readily available. Rodents who had learned helplessness showed inhibited firing of cells in the ventral tegmental area, less activity in the nucleus accumbens, and apparent anhedonia. This inhibition was mediated via messages from the infralimbic prefrontal cortex (the equivalent to the subgenual cingulate cortex in humans, important for motivation) to the amygdala and then the GABAergic ventral pallidum, which decreased the number of dopaminergic cells firing in the ventral tegmental area. Blocking the amygdala input to this inhibitory pathway reversed the low dopamine firing and the anhedonia-like behaviors.
The anesthetic ketamine (which has rapid-acting antidepressant effects in humans) produces an immediate reversal of the learned helpless behavior in the rodents and increases the number of dopamine cells firing in the ventral tegmental area. Ketamine administered directly into the nucleus accumbens induces long-term potentiation (enhanced synaptic responsivity) and reverses helpless behavior and the long-term depression of neural firing that is associated with it.
Thus, when an acute stressor is over and the opponent process emerges, or following long-term chronic stressors such as learned helplessness, the excitatory path to the ventral tegmental area is absent, while the inhibitory path to the ventral tegmental area (via the infralimbic prefrontal cortex, amygdala, and ventral pallidum) predominates. Ketamine is able to re-activate the activating pathway and increase activity in the ventral tegmental area and the nucleus accumbens, changes that are associated with the reversal of learned helplessness and anhedonia.
Editor’s Note: In the previous BNN, we reported researcher Scott Russo’s findings that input from the intralaminar nucleus of the thalamus was critical to the depression-like behaviors seen in a different animal model of depression, social defeat stress, where repeated exposure to defeat by a larger, more aggressive animal produces behaviors that resemble human depression. Here in Pitts’ research, learned helplessness is induced by inescapable shocks. Both models share the finding that firing decreases in the reward area of the brain (the nucleus accumbens). However, the key part of the brain driving the low levels of activity in the nucleus accumbens and the associated depression-like behavior appear to be different in these two different models. The intralaminar nucleus of the thalamus plays a key role in the social defeat stress model, while the infralimbic cortex and the amygdala play key roles in the learned helplessness model. These data together suggest that part of the reason depression differs from person to person may be because the illness can be driven by different brain areas as a result of different kinds of stressors.
To study depression in humans, researchers look to rodents to learn more about behavior. Rodents who are repeatedly defeated by more aggressive animals often begin to exhibit behavior that resembles depression. At the 2014 meeting of the International College of Neuropsychopharmacology (CINP), researcher Andre Der-Avakian reported that in a recent study, repeated experiences of social defeat led to depressive behavior in a subgroup of animals (which he calls susceptible), but not in others (which he calls resilient). Among many biological differences, the resilient animals showed increases in neurogenesis in the dentate gyrus of the hippocampus.
Chronic treatment of the susceptible animals with the selective serotonin reuptake inhibitor (SSRI) antidepressant fluoxetine or the tricyclic antidepressant desipramine, which both increase neurogenesis, also reversed the depressive behavior in about half of the animals. A single injection of the anesthetic ketamine (which has rapid-acting antidepressant effects in humans) reversed social avoidance behavior in about 25% of the animals. One depression-like symptom was anhedonia (loss of pleasure from previously enjoyed activities), which researchers measured by observing to what extent the animals engaged in intracranial self-stimulation, pressing a bar to stimulate the brain pleasurably. The effectiveness of the drugs in inducing resilient behavior was related to the degree of anhedonia seen in the animals. The drugs worked less well in the more anhedonic animals (those who gave up the intracranial stimulation more easily, indicating that they experienced less reward from it.)
Raphael Mechoulam, who first synthesized THC, the main ingredient in marijuana, gave the history of marijuana and its receptors in the central nervous system in a plenary talk at the 2014 meeting of the International College of Neuropsychopharmacology. In Syria hundreds of years ago the drug was named ganzigunnu, meaning “the drug that takes away the mind.” It has also been called azalla, meaning “hand of the ghost.” Among the 100 compounds in marijuana, the best-known ingredient is delta-9-tetrahydrocannabinol (delta-9 THC), which produces most of the actions of the drug. There is another active ingredient, cannabidiol (CBD), which has calming and anti-anxiety effects, but is present in very low levels.
The brain has cannabinoid receptors that respond to ingredients in marijuana in addition to other chemicals produced in the brain. They modulate calcium ions and decrease the release of many neurotransmitters.
THC acts at CB-1 receptors, producing the high. The CB-1 receptor is synthesized on demand, post-synaptically, and is transferred to the pre-synaptic terminal where it decreases calcium and transmitter release. Consistent with marijuana’s appetite-stimulating properties (“the munchies”), if the CB-1 receptor is blocked in animals, they lose their appetite and die of hunger.
There are also low levels of CB-2 receptors in the brain, whose activation does not cause a high, and whose levels may increase dramatically in pathological situations. Activation of the CB-2 receptor is anti-inflammatory and, in the same way that the immune system acts against foreign proteins, CB-2 acts as a protector against non-proteins.
CBD does not bind to any cannabinoid receptors, but its actions are blocked by cannabinoid antagonists.
There are two chemicals in the brain (endogenous ligands) that act at cannabinoid receptors—anandamide and 2-arachidonoylglycerol (2-AG). They are soluble only in lipids (not in water), and have never been given to people. In animals, 2-AG has neuroprotective effects, decreases the size of a stroke by 60%, and increases recovery from stroke.
Marijuana and CBD in particular have also had beneficial effects in people. Marijuana decreases the nausea and vomiting associated with chemotherapy in children, has anti-inflammatory effects in rheumatoid arthritis (decreasing inflammatory marker TNF alpha), and has anti-diabetes and anti-convulsant effects.
In 2012, researcher F. Markus Leweke and colleagues showed that CBD was about as effective as the atypical antipsychotic amisulpiride in alleviating the psychotic symptoms of schizophrenia. CBD’s other effects include reducing anxiety and improving psoriasis by increasing DNA methylation (Pucci et al. 2013).
It seems possible that some of these myriad effects of marijuana and endogenous ligands at CB receptors could be exploited for clinical therapeutics, as Mechoulam endorses, but when and how that will take place remains an unanswered question.
Editor’s Note: Despite all these potential positives of CBD, it should be noted that its levels are very low in marijuana, and that heavy smoking of marijuana has substantial adverse effects. These include low motivation, a doubling of the risk of psychosis, a hastening of the onset of bipolar disorder and schizophrenia, and cognitive impairment, as well as some changes in brain structure seen via magnetic resonance imaging (MRI). It may be becoming legal in many states, but is a bad idea for those at high risk for mood, anxiety, or bipolar disorders or for schizophrenia.
At the 2014 meeting of the International College of Neuropsychopharmacology, researcher Joseph Zohar presented a poster on the effects of early post-stressor intervention with the drug agomelatine in animals who showed behavioral and molecular responses to stress that served as a model of post-traumatic stress disorder (PTSD).
Agomelatine is available clinically as an antidepressant in Canada and Europe (but not in the US), and can also reduce anxiety and re-synchronize circadian rhythms. Agomelatine is a melatonin (MT1/MT2) receptor agonist and a serotonin 5HT2C antagonist (increasing dopamine and norepinephrine in the frontal cortex).
Long-term behavioral, molecular and structural effects of the drug were assessed in animals. Adult male Sprague-Dawley rats were exposed to the scent of a predator for 10 minutes, and one hour later they were treated acutely for this stress with agomelatine (50mg/kg i.p.) or placebo.
Agomelatine decreased the prevalence of extreme, PTSD-like behavioral and molecular responses to the stressor, such as freezing in place and increased corticosterone. Agomelatine also normalized decreases in brain-derived neurotrophic factor (BDNF) observed in the dentate gyrus of the hippocampus, the cortex (layer III), and the basolateral amygdala. In line with this, agomelatine-treated stressed animals displayed significantly increased number and length of dendrites at glutamate synapses in the hippocampus (including the dentate gyrus and CA1) and reversed the hippocampal neuronal retraction observed in the rats who were given the placebo.
Agomelatine also affected the expression of clock genes in the rats, which regulate biorhythms. These genes lead to the production of the major clock gene proteins Per1 and Per2. Agomelatine normalized Per1 increases in three parts of the brain: the CA3, another glutamate synapse near the dentate gyrus; the suprachiasmatic nucleus over the optic chiasm, important for circadian rhythms; and the basolateral amygdala. Per2, a protein that also drives circadian rhythms, increased in the CA1 synapse of the hippocampus, the suprachiasmatic nucleus and the basolateral amygdala of the stressed rats.
The researchers concluded that the data provide “initial evidence that a single dose of agomelatine administered in the acute aftermath of stress promotes recovery while promoting enhanced neuronal and synaptic plasticity and connectivity in the secondary prevention of PTSD in this model.”
Patients with Schizophrenia Have Short Telomeres, Atypical Antipsychotics Increased Telomere Length in Animals
Telomeres sit at the end of DNA strands and shorten with each cell replication. A person’s percentage of short telomeres increases with aging. Telomeres also shorten with childhood adversity and as a function of number of depressive episodes.
In a recent study, researcher Kazuya Torimi found that telomeres in white cells were shorter in 42 patients with schizophrenia compared to 56 healthy control participants.
Torimi also treated mice with various antipsychotics for 2-week periods. Treatment with atypical antipsychotics, such as risperidone, olanzapine and aripiprazole, but not typical antipsychotics like haloperidol, elongated telomere length in the hippocampus. This probably occurred through effects on serotonin.
Torimi suggests that atypical antipsychotics improve negative symptoms in part through the modulation of telomere length.
Editor’s Note: Lithium has been found to increase telomere length in patients with bipolar disorder, and appears to do this through a direct effect on telomerase, an enzyme responsible for adding to telomere length. Short telomeres are associated with a large number of medical and psychiatric illnesses.
The antidepressant agomelatine (which is available in many countries, but not the US) and the anti-insomnia drug ramelteon (Rozerem) both act as agonists at melatonin M1 and M2 receptors. New research is clarifying the role of these receptors in sleep.
In new research from Stefano Comai et al., mice who were genetically altered to have no M1 receptor (MT1KO knockout mice) showed a decrease in rapid eye movement (REM) sleep, which is linked to dreaming, and an increase in slow wave sleep. Mice who were missing the M2 receptor (MT2KO knockout mice) showed a decrease in slow wave sleep. The effects of knocking out a particular gene like M1 or M2 end up being opposite to the effect of stimulating the corresponding receptor.
The researchers concluded that MT1 receptors are responsible for REM sleep (increasing it while decreasing slow wave sleep), and MT2 receptors are responsible for slow wave non-REM sleep.
The new information about these melatonin receptors may explain why oral melatonin supplements can make a patient fall asleep faster, but do not affect the duration of non-REM sleep. The authors suggest that targeting MT2 receptors could lead to longer sleep by increasing slow wave sleep, potentially helping patients with insomnia.
Autism spectrum disorders are associated with developmental abnormalities at excitatory synapses. Dendrites, the branched projections of neurons where electrical signals are passed from one cell to the next, are covered in hundreds to thousands of spines that facilitate the synaptic connections with other neurons. These spines are created and also pruned as part of normal learning and development.
Post-mortem examination of the brains of patients with autism spectrum disorders shows increased density of dendritic spines and less pruning in certain neurons in the temporal lobe. These examinations also show impaired mTOR autophagy. MTOR is a protein that plays a role in cell growth and survival. Autophagy is the normal process by which some components of cells are broken down.
A 2014 study by Guomei Tang et al. in the journal Neuron showed that mice that are genetically altered to have overactive mTOR also have reduced dendritic spine pruning, blockade of autophagy, and increased autism-like behaviors. An immunosuppressant drug called rapamycin inhibits mTOR, and treating the mice with this drug corrected the problems with spine pruning and the autism-like behaviors. (This was not true for mice who had been altered to have another type of autophagy.) Normal spine formation was not affected by the restored pruning ability.
Tang et al. concluded that mTOR autophagy plays an important role in dendritic spine pruning, and that restoring neuronal autophagy can correct synaptic abnormalities and restore normative social behavior in mice with hyperactive mTOR.
At the 2014 meeting of the International College of Neuropsychopharmacology, researcher Scott Russo described characteristics of rodents who showed depression-like behavior after 10 days of exposure to a larger, more aggressive animal (a phenomenon known as defeat stress). These animals exhibited many behaviors that resembled human depression, including anxiety-like behaviors while navigating a maze; activation of the hypothalamic-pituitary-adrenal axis; circadian rhythm abnormalities; metabolic changes such as glucose intolerance; susceptibility to addiction; anhedonia, a lack of interest in sucrose, sex or intracranial self-stimulation; and profound and permanent social avoidance.
In susceptible animals, Russo found anatomical changes in the GABAergic neurons of the nucleus accumbens (also known as the ventral striatum), including increased numbers of synapses and a greater number of stubby spines on dendrites (the branched projections of neurons where electrical signals are passed from one cell to the next), as well as greater excitability of glutamatergic input, observed as excitatory post-synaptic potentials.
Russo’s attempt to identify these key neurons among the billions of neurons and the 100 to 500 trillion synapses in the brain was like the search for a needle in a haystack, but thinks he found it. The medium spiny neurons of the nucleus accumbens contain GABA and receive synapses from the prefrontal cortex, amygdala, and intralaminar nucleus of the thalamus (ILT), in addition to dopamine inputs from the VTA, and cholinergic, somatostatin, and orexin inputs. Russo found that it was the ILT inputs that conveyed susceptibility to defeat stress, and their presynaptic endings showed increased levels of glutamate transporters (VGLUT-2). Driving the ILT was sufficient to cause the rodents to display the depression-like behaviors, and silencing the ILT during defeat stress prevented the susceptible behaviors (like social avoidance) and promoted resilience.
Epidemiological studies have linked methamphetamine use to risk of Parkinson’s disease, and animal studies of the illicit drug have shown that it harms dopamine neurons. A 2014 study by Sara Ares-Santos et al. in the journal Neuropsychopharmacology compared the effects of repeated low or medium doses to those of a single high dose on mice. Loss of dopaminergic terminals, where dopamine is released, was greatest after three injections of 10mg/kg given at three-hour intervals, followed by three injections of 5 mg/kg at three-hour intervals, and a one-time dose of 30mg/kg. All of the dosages produced similar rates of degeneration of dopamine neurons via necrosis (cell destruction) and apoptosis (cell suicide) in the substantia nigra pars compacta (the part of the brain that degenerates in Parkinson’s disease) and the striata.