Directional summation in non-direction selective retinal ganglion cells.

Retinal ganglion cells receive inputs from multiple bipolar cells which must be integrated before a decision to fire is made. Theoretical studies have provided clues about how this integration is accomplished but have not directly determined the rules regulating summation of closely timed inputs along single or multiple dendrites. Here we have examined dendritic summation of multiple inputs along On ganglion cell dendrites in whole mount rat retina. We activated inputs at targeted locations by uncaging glutamate sequentially to generate apparent motion along On ganglion cell dendrites in whole mount retina. Summation was directional and dependent13 on input sequence. Input moving away from the soma (centrifugal) resulted in supralinear summation, while activation sequences moving toward the soma (centripetal) were linear. Enhanced summation for centrifugal activation was robust as it was also observed in cultured retinal ganglion cells. This directional summation was dependent on hyperpolarization activated cyclic nucleotide-gated (HCN) channels as blockade with ZD7288 eliminated directionality. A computational model confirms that activation of HCN channels can override a preference for centripetal summation expected from cell anatomy. This type of direction selectivity could play a role in coding movement similar to the axial selectivity seen in locust ganglion cells which detect looming stimuli. More generally, these results suggest that non-directional retinal ganglion cells can discriminate between input sequences independent of the retina network.

PIP2-mediated HCN3 channel gating is crucial for rhythmic burst firing in thalamic intergeniculate leaflet neurons.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate a pacemaking current, I(h), which regulates neuronal excitability and oscillatory activity in the brain. Although all four HCN isoforms are expressed in the brain, the functional contribution of HCN3 is unknown. Using immunohistochemistry, confocal microscopy, and whole-cell patch-clamp recording techniques, we investigated HCN3 function in thalamic intergeniculate leaflet (IGL) neurons, as HCN3 is reportedly preferentially expressed in these cells. We observed that I(h) recorded from IGL, but not ventral geniculate nucleus, neurons in HCN2(+/+) mice and rats activated slowly and were cAMP insensitive, which are hallmarks of HCN3 channels. We also observed strong immunolabeling for HCN3, with no labeling for HCN1 and HCN4, and only very weak labeling for HCN2. Deletion of HCN2 did not alter I(h) characteristics in mouse IGL neurons. These data together indicate that the HCN3 channel isoform generated I(h) in IGL neurons. Intracellular phosphatidylinositol-4,5-bisphosphate (PIP(2)) shifted I(h) activation to more depolarized potentials and accelerated activation kinetics. Upregulation of HCN3 function by PIP(2) augmented low-threshold burst firing and spontaneous oscillations; conversely, depletion of PIP(2) or pharmacologic block of I(h) resulted in a profound inhibition of excitability. The results indicate that functional expression of HCN3 channels in IGL neurons is crucial for intrinsic excitability and rhythmic burst firing, and PIP(2) serves as a powerful modulator of I(h)-dependent properties via an effect on HCN3 channel gating. Since the IGL is a major input to the suprachiasmatic nucleus, regulation of pacemaking function by PIP(2) in the IGL may influence sleep and circadian rhythms.

Dendritic HCN2 channels constrain glutamate-driven excitability in reticular thalamic neurons.

Hyperpolarization activated cyclic nucleotide (HCN) gated channels conduct a current, I(h); how I(h) influences excitability and spike firing depends primarily on channel distribution in subcellular compartments. For example, dendritic expression of HCN1 normalizes somatic voltage responses and spike output in hippocampal and cortical neurons. We reported previously that HCN2 is predominantly expressed in dendritic spines in reticular thalamic nucleus (RTN) neurons, but the functional impact of such nonsomatic HCN2 expression remains unknown. We examined the role of HCN2 expression in regulating RTN excitability and GABAergic output from RTN to thalamocortical relay neurons using wild-type and HCN2 knock-out mice. Pharmacological blockade of I(h) significantly increased spike firing in RTN neurons and large spontaneous IPSC frequency in relay neurons; conversely, pharmacological enhancement of HCN channel function decreased spontaneous IPSC frequency. HCN2 deletion abolished I(h) in RTN neurons and significantly decreased sensitivity to 8-bromo-cAMP and lamotrigine. Recapitulating the effects of I(h) block, HCN2 deletion increased both temporal summation of EPSPs in RTN neurons as well as GABAergic output to postsynaptic relay neurons. The enhanced excitability of RTN neurons after I(h) block required activation of ionotropic glutamate receptors; consistent with this was the colocalization of HCN2 and glutamate receptor 4 subunit immunoreactivities in dendritic spines of RTN neurons. The results indicate that, in mouse RTN neurons, HCN2 is the primary functional isoform underlying I(h) and expression of HCN2 constrains excitatory synaptic integration.

Antagonist-induced increase in 5-HT1A-receptor expression in adult rat hippocampus and cortex.

Many receptor antagonists function as reverse agonists on the signaling transduction pathway, but little is known about the action of these drugs on the regulation of receptor expression. Serotonin 1A (5-HT1A) receptor expression in 5-HT and serum-free fetal hippocampal cultures is increased in the presence of a specific 5-HT1A-receptor antagonist N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635). To study the plasticity of postsynaptic 5-HT1A receptors in the presence of antagonist in vivo, adult Sprague Dawley rats were injected i.p. either once or twice daily with a dose of WAY 100635 (3 mg/kg) over a period of 3 days. The 5-HT1A receptor expression was detected by immunocytochemistry and light microscopy, and the receptor immunoreactivity (IR) in hippocampus subregions was quantitatively assessed by using a comparative computer-assisted morphometric analysis. Following the daily injections of WAY 100635, a significant increase in 5-HT1A receptor labeling in hippocampal neurons was recorded. This marked increase in 5-HT1A receptor expression, which occurred within 4 h after a single injection of WAY 100635, is evident on the somata membrane and dendritic processes of hippocampal and cortex layer V neurons. By contrast, no increase in 5-HT1A receptor-IR was observed after multiple daily injections at a low dose (1 mg/kg) of WAY 100635. Our study shows that a single or multiple daily injections of WAY 100635 can result in an increase in 5-HT1A receptor-IR. This increase in labeling is consistent with an enhanced expression of the receptor protein. The action of this "inverse agonist" may have clinical importance in disorders such as depression, epilepsy, and Alzheimer's disease in which 5-HT1A receptor levels are deficient.

Propofol block of I(h) contributes to the suppression of neuronal excitability and rhythmic burst firing in thalamocortical neurons.

Although the depressant effects of the general anesthetic propofol on thalamocortical relay neurons clearly involve gamma-aminobutyric acid (GABA)(A) receptors, other mechanisms may be involved. The hyperpolarization-activated cation current (I(h)) regulates excitability and rhythmic firing in thalamocortical relay neurons in the ventrobasal (VB) complex of the thalamus. Here we investigated the effects of propofol on I(h)-related function in vitro and in vivo. In whole-cell current-clamp recordings from VB neurons in mouse (P23-35) brain slices, propofol markedly reduced the voltage sag and low-threshold rebound excitation that are characteristic of the activation of I(h). In whole-cell voltage-clamp recordings, propofol suppressed the I(h) conductance and slowed the kinetics of activation. The block of I(h) by propofol was associated with decreased regularity and frequency of delta-oscillations in VB neurons. The principal source of the I(h) current in these neurons is the hyperpolarization-activated cyclic nucleotide-gated (HCN) type 2 channel. In human embryonic kidney (HEK)293 cells expressing recombinant mouse HCN2 channels, propofol decreased I(h) and slowed the rate of channel activation. We also investigated whether propofol might have persistent effects on thalamic excitability in the mouse. Three hours following an injection of propofol sufficient to produce loss-of-righting reflex in mice (P35), I(h) was decreased, and this was accompanied by a corresponding decrease in HCN2 and HCN4 immunoreactivity in thalamocortical neurons in vivo. These results suggest that suppression of I(h) may contribute to the inhibition of thalamocortical activity during propofol anesthesia. Longer-term effects represent a novel form of propofol-mediated regulation of I(h).