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Summary: Steven Siegelbaum's laboratory studies mechanisms underlying the regulation of synaptic transmission and neuronal integration that are important for learning and memory, focusing on the hyperpolarization-activated channels present in neuronal dendrites.

There is now strong evidence that learning and memory depend on long-term changes in the strength of synaptic transmission between neurons. However, a typical neuron receives 10,000 synaptic inputs that are distributed along complex dendritic processes emanating from the neuron's cell body. This synaptic information must be integrated by the neuron to determine whether it will fire an action potential, the output signal. It is now clear that neuronal dendrites are not passive recipients of synaptic information but active processors of this information to regulate neural function. Although the role of long-term synaptic plasticity in learning and memory has been widely studied, we know much less about the contribution of active dendritic processing to information storage.

Our laboratory has focused on the role of the hyperpolarization-activated channels—present in many neuronal dendrites—in regulating synaptic integration and long-term plasticity during learning and memory. These channels are encoded by a family of four genes, HCN1–4 (so named because the channels are hyperpolarization-activated, cation nonselective, and regulated by cyclic nucleotide binding). The HCN1 channel is expressed heavily in specific brain regions important for learning and memory, including the hippocampus, and is present in neuronal dendrites. In collaboration with Eric Kandel's laboratory (HHMI, Columbia University College of Physicians and Surgeons), we have been studying the importance of these channels for learning and memory by analyzing mice in which the HCN1 gene has been deleted through homologous recombination.

	Sites of evoked transmitter release in hippocampal neurons...

We previously reported an unexpected result. Mice in which the HCN1 gene has been selectively deleted from the forebrain, which includes the hippocampus, show an enhancement in their ability to learn to navigate to a hidden platform in a water maze. Why does deletion of HCN1 in the forebrain improve spatial learning? To answer this question we are studying synaptic transmission and long-term synaptic plasticity in pyramidal neurons from the CA1 region of the hippocampus, the neurons that provide the major output of the hippocampus. These neurons receive two major sources of excitatory synaptic input. One set of inputs, the Schaffer collaterals, come from hippocampal CA3 pyramidal neurons and terminate on regions of CA1 neuron dendrites that are relatively close to the cell body, an area with only moderate HCN1 expression. The second set of inputs, the perforant path, represent a direct connection from the neocortex and terminate on the distal dendritic regions of the CA1 neurons, an area where HCN1 expression is very high.

Synaptic transmission and long-term plasticity at the more proximal Schaffer collateral pathway are relatively unaffected by the deletion of HCN1. In contrast, we observed a significant enhancement in the integration of synaptic potentials and a striking increase in long-term potentiation (LTP) of synaptic transmission at the distal perforant path synapses. The differential effect of HCN1 deletion on the two synaptic inputs is consistent with the normal pattern of HCN1 expression in the dendrites of wild-type mice.

Why does HCN1 deletion enhance the induction of LTP at the perforant path synapses? Previous studies from other labs suggest that induction of LTP requires the firing of local calcium action potentials in the distal dendrites. We therefore have recently imaged calcium in distal dendrites from wild-type and HCN1-knockout mice during stimulation of the perforant path inputs. Brief bursts of synaptic activity elicit large, long-lasting calcium signals that last on average 200–300 milliseconds, far longer than the 100-millisecond period of synaptic stimulation. In mice with a deletion of HCN1, the same synaptic stimulation induces a calcium spike with a twofold increase in duration, compared to the calcium signal in wild-type mice. We see a similar enhancement in the calcium signal using a pharmacological antagonist of HCN channel function.

The inhibitory effects of HCN channels are somewhat puzzling since the channels generate a depolarizing current that should help excite a neuron. Our results suggest that this paradoxical inhibitory effect is due to the fact that HCN channels help make the resting potential of a neuron more positive. Because the more positive resting potential closes the inactivation gates of voltage-gated sodium and calcium channels, the availability of these channels to participate in an action potential is decreased.

These results show that HCN1 selectively regulates synaptic transmission, plasticity, and dendritic calcium signals at the distal perforant path synapses, with less effect on more proximal Schaffer collateral synapses. This differential action reflects the subcellular pattern of HCN1 expression. Antibody staining shows a gradient of increasing HCN1 expression in CA1 dendrites with increasing distance from the cell body. At the most distal dendrites, the density of HCN1 expression is 60 times that in the cell body. How is this gradient established? What proteins are involved in HCN1 trafficking? These questions are clinically relevant since down-regulation of HCN1 occurs during seizures and may contribute to the development of epilepsy.

To address the molecular mechanisms that regulate HCN channel trafficking, we previously performed a yeast two-hybrid screen to isolate proteins that interact with the HCN channel family. We identified TRIP8b, a cytoplasmic protein that specifically binds to the extreme carboxyl terminus of the HCN channels, including HCN1. TRIP8b shows a dendritic pattern of expression that is similar to that of HCN1, and deletion of HCN1 alters TRIP8b expression, indicating that the two proteins interact in vivo. When TRIP8b is coexpressed with HCN1 in cell lines or overexpressed in a hippocampal CA1 neuron, it leads to a dramatic down-regulation in surface expression of HCN1. More recently we have found that TRIP8b is alternatively spliced at its amino terminus. A second TRIP8b isoform has the opposite effect on HCN1 trafficking, leading to a significant increase in channel expression. We are examining the relative importance of these different splice variants in vivo.

These results help define the functional roles and targeting mechanisms for the high levels of expression of HCN1 in distal dendrites of CA1 pyramidal neurons. They also support the idea that neurons utilize elaborate mechanisms to control information processing differentially at distinct sites within their dendritic tree. Moreover, the marked effects of HCN1 deletion on spatial learning and memory indicate that such dendritic processing is likely to be a key event in the proper functioning of neural circuits.

Last updated: December 5, 2007