NEUROBIOLOGY

Toward Curbing Enthusiasm

Regulator Found that Controls Formation of Inhibitory Synapses on Excitatory Neurons

The power of positive influence attracts biologists as readily as it does the viewers of Oprah, the followers of megachurch pastors, and the readers of self-help books. Studies of activators naturally precede those of repressors. Likewise, in the brain, scientists have focused on the predominant excitatory synapses to learn how sensations, activities, and ideas sculpt a vast network of neurons just after birth and refresh them throughout life.

In a new paper, researchers report how the same sensory experiences also install a crucial negative feedback system. The findings eventually may provide insights into autism, schizophrenia, and epilepsy by opening up the field of study of inhibitory synapses, which presumably help regulate the unrestrained exuberance of excitatory impulses.

Graham Ramsay

From left, Michael Greenberg, Yingxi Lin, Brenda Bloodgood, and their co-authors discovered a molecule that forms and sustains inhibitory synapses in response to excitatory nerve impulses.

“The control of the excitatory–inhibitory balance may be at the heart of disorders such as autism, but the mechanisms involved are mostly unknown,” said senior author Michael Greenberg, director of the Neurobiology Program at Children’s Hospital Boston and HMS neurobiology chair. “We have found the first transcriptional regulator that controls the formation of inhibitory synapses on excitatory neurons.”

“This study brings inhibitory neurons and synapses to the front stage,” said Josh Huang, professor of neuroscience at Cold Spring Harbor Laboratory, who was not involved in the study. “One goal of neuroscience is to understand how experience in the form of neuronal activity regulates the assembly and modification of neural circuitry, where our brain function is embedded. Circuits consist of excitatory and inhibitory neurons and synapses, but for a long time, the tools and understanding of inhibitory neurons have been lagging.”

The Editors
Inhibitory neurons and their connections make up fewer than 10 percent of the estimated trillions of neurons and quadrillions of synapses. They talk in the language of GABA, an inhibitory neurotransmitter.

The calming neurons come in more than a dozen colorful personalities, varying in appearance, molecular markers, and functional properties. In connecting to other neurons, highly customized GABAergic axons strategically target sites of more centralized control—cell bodies, dendritic shafts, and occasional axons. In contrast, the more homogeneous excitatory neurons release glutamate mostly at synapses on dendritic spines.

“I like to think of excitatory inputs as the information superhighway,” said first author Yingxi Lin. “That’s the bulk of the information. Inhibitory neurons are more like the gatekeepers that decide what goes through. They have distinctive firing patterns so they can modulate the output of the circuit with punctuation and spacing.”

Evidence is building for the importance of GABA in normal brain plasticity and neurological disorders. Several years ago, for example, Takao Hensch, also at Children’s and not involved in this study, found an unexpected requirement of GABA synapses for the correct opening and closing of critical periods (windows of heightened sensitivity to certain environmental stimuli that evoke transformations in the brain that would be difficult or impossible at other times).

All this was in the back of Lin’s mind when she launched the study six years ago. “Neurons need both positive and negative feedback to create certain patterns of activity,” she said. “If the building blocks are all positive, it would be harder to come up with a flexible and stable circuit. With both positive and negative, the circuit can generate complicated and precise spatial and temporal patterns in the brain.”

Lin started by applying the relatively new DNA microarray technology to look for genes regulated by neuronal activity. She found more than 300 genes turned up or down in a neuron culture mimicking excitatory inputs. To avoid a labor-intensive gene-by-gene evaluation, she proposed three guiding criteria to help narrow important candidates for further study. The first, that the genes be activity-regulated, was met by the screen.

Next, Lin reasoned, the candidate genes should function postsynaptically. “I feel the neuron needs to decide how many excitatory and inhibitory synapses it needs,” said Lin, now an assistant professor of neuroscience at the McGovern Institute for Brain Research at MIT. “The neurons sending activity to another neuron don’t decide how much information it needs. It receives so many inputs and has to integrate all that information and decide if it all is too much. The decision has to come from within. It has to be postsynaptic.”

Finally, Lin wanted a transcription regulator that worked like a master switch to set in motion the cascade of events necessary for long-term changes in the neuron anticipated for learning and memory.

To identify a specific player and weed out more generally responsive genes, Lin ran a secondary screen to find genes selectively activated by calcium influx, the mediator of neurotransmitter signaling and gene activity. Only one gene, named Npas4, made the final cut.

Inhibitory Architect
The first phase of subsequent experiments showed a neuron needs Npas4, a transcription factor, to either form or sustain inhibitory synapses. In a survey of other tissues, Lin and her co-authors soon determined, the transcription factor seemed to be expressed only in the brain. Knocking down the gene using RNA interference in single neurons decreased the number of inhibitory synapses. Increasing the amount of Npas4 boosted GABA synapses.

The second phase showed how Npas4 maintains inhibitory synapses in homeostasis. For this phase, Lin enlisted postdoctoral colleague Brenda Bloodgood, second author on the paper, to take electrophysiology recordings of neurons with too little or too much Npas4. The circuit was likely to be correspondingly overexcited or overinhibited.

Connect the dots. The number of GABAergic synapses is greatly reduced in cultured mouse hippocampal neurons where Npas4 has been knocked down by RNAi (top right) compared to control neurons (bottom right). A synapse on the cell body or dendrite is counted when a blue-stained presynaptic synapse overlaps with the red-stained postsynaptic synapse. This experiment and others defined the transcription factor as a crucial regulatory control for making or maintaining inhibitory synapses on excitatory cells.

Follow-up studies in animals need to be conducted to learn what actually happens in a brain, the authors say. Thanks to their research, they and others now have a way to study inhibitory synapses by investigating the impact and influence of Npas4. A neuronal circuit’s loss of Npas4 affects at least 300 genes, Lin and her co-authors found in another microarray experiment. One of those, BDNF, is perhaps the best known molecular player in the GABA synapse, in part due to previous studies by Huang and others. Npas4 acted in part on BDNF to attenuate inhibitory synapses, but also on other as-yet unidentified downstream players, further experiments in neuron cultures showed.

Meanwhile, Npas4 appears to regulate one of the genes connected to some cases of autism in a genetic study from the lab of Christopher A. Walsh, chief of genetics at Children’s (see story in the July 11 issue of Focus). The Greenberg study was published online Sept. 24 in Nature.

—Carol Cruzan Morton

Conflict Disclosure: No conflicts declared

Sources of Funding: F.M. Kirby Foundation, the Nancy Lurie Marks Family Foundation, the Lefler Foundation, the National Institutes of Health, Ruth L. Kirschstein National Research Service Award, and Helen Hay Whitney Foundation Postdoctoral Fellowship.

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