Enhanced GABAA-Mediated Tonic Inhibition in Auditory Thalamus of Rats with Behavioral Evidence of Tinnitus.

Accumulating evidence suggests a role for inhibitory neurotransmitter dysfunction in the pathology of tinnitus. Opposing hypotheses proposed either a pathologic decrease or increase of GABAergic inhibition in medial geniculate body (MGB). In thalamus, GABA mediates fast synaptic inhibition via synaptic GABAA receptors (GABAARs) and persistent tonic inhibition via high-affinity extrasynaptic GABAARs. Given that extrasynaptic GABAARs control the firing mode of thalamocortical neurons, we examined tonic GABAAR currents in MGB neurons in vitro, using the following three groups of adult rats: unexposed control (Ctrl); sound exposed with behavioral evidence of tinnitus (Tin); and sound exposed with no behavioral evidence of tinnitus (Non-T). Tonic GABAAR currents were evoked using the selective agonist gaboxadol. Months after a tinnitus-inducing sound exposure, gaboxadol-evoked tonic GABAAR currents showed significant tinnitus-related increases contralateral to the sound exposure. In situ hybridization studies found increased mRNA levels for GABAAR δ-subunits contralateral to the sound exposure. Tin rats showed significant increases in the number of spikes per burst evoked using suprathreshold-injected current steps. In summary, we found little evidence of tinnitus-related decreases in GABAergic neurotransmission. Tinnitus and chronic pain may reflect thalamocortical dysrhythmia, which results from abnormal theta-range resonant interactions between thalamus and cortex, due to neuronal hyperpolarization and the initiation of low-threshold calcium spike bursts (Walton and Llinás, 2010). In agreement with this hypothesis, we found tinnitus-related increases in tonic extrasynaptic GABAAR currents, in action potentials/evoked bursts, and in GABAAR δ-subunit gene expression. These tinnitus-related changes in GABAergic function may be markers for tinnitus pathology in the MGB.

Responsiveness to nicotine of neurons of the caudal nucleus of the solitary tract correlates with the neuronal projection target.

The caudal nucleus of the solitary tract (NTS) is the key integrating center of visceral sensory-motor signaling supporting autonomic homeostasis. Two key projections of this nucleus are the parabrachial nucleus (PbN) and the dorsal motor nucleus of the vagus (DMV). The PbN integrates and relays viscerosensory information primarily to the forebrain, supporting behavioral, emotional, and endocrine responses to visceral events, while the DMV contains parasympathetic preganglionic cholinergic motoneurons that support primarily gastrointestinal reflexes. Subsets of caudal NTS neurons express presynaptic and somatodendritic nicotinic acetylcholine receptors (nAChRs). However, the anatomical identification of nicotine-responsive caudal NTS neurons has not been determined. This study used in vivo and ex vivo fluorescent tracing and slice patch-clamp electrophysiological recordings from anatomically identified caudal NTS neurons to test the hypothesis that the responsiveness of these cells to nicotine correlates with the target of their axonal projections. The results demonstrate that the majority of glutamatergic terminals that synapse on PbN-projecting caudal NTS neurons are unaffected by nicotine. Moreover, only a fraction of these cells express somatodendritic nAChRs. In contrast, the majority of DMV-projecting caudal NTS neurons exhibit robust presynaptic and somatodendritic responsiveness to nicotine. However, PbN-projecting neurons also exhibit significantly lower background frequencies of glutamatergic miniature postsynaptic currents than DMV-projecting neurons. Therefore, presynaptic unresponsiveness to nicotine may result from deficient glutamatergic innervation of PbN-projecting neurons. Nevertheless, the caudal NTS contains function-specific subsets of cells with target-specific responsiveness to nicotine. These results may support development of therapeutic strategies for selective targeting of specific autonomic pathways and impaired autonomic homeostasis.

Synaptic strength and postsynaptically silent synapses through advanced aging in rat hippocampal CA1 pyramidal neurons.

Synaptic dysfunction is thought to contribute to age-related learning impairments. Detailed information regarding the presence of silent synapses and the strength of functional ones through advanced aging, however, is lacking. Here we used paired-pulse minimal stimulation techniques in CA1 stratum radiatum to determine whether the amplitude of spontaneous and evoked miniature excitatory postsynaptic currents (sEPSCs and eEPSCs, respectively) changes over the lifespan of rats in hippocampal CA1 pyramidal neurons, and whether silent synapses are present in adult and aged rats. The amplitudes of both sEPSCs and eEPSCs at resting membrane potential (i.e., clamped at -65 mV) initially increased between 2 weeks and 3 months, but then remained constant through 36 months of age. The potency of the eEPSCs at depolarized membrane potentials (i.e., clamped at +40 mV), however, was highest among 36-month old rats. Additionally, presynaptically silent synapses in CA1 stratum radiatum disappeared between 2 weeks and 3 months, but postsynaptically silent synapses were present through advanced aging. The similarity of silent and functional synapses in CA1 hippocampus at resting membrane potentials throughout adulthood in rats may indicate that impairments in the mechanisms of synaptic plasticity and its subsequent stabilization, rather than deficient synaptic transmission, underlie age-related cognitive decline. Such a notion is consistent with the increased amplitude of synaptic currents at depolarized potentials, perhaps suggesting an upregulation in the expression of synaptic NMDA receptors once rats reach advanced age.

Balanced gene regulation by an embryonic brain ncRNA is critical for adult hippocampal GABA circuitry.

Genomic studies demonstrate that, although the majority of the mammalian genome is transcribed, only about 2% of these transcripts are code for proteins. We investigated how the long, polyadenylated Evf2 noncoding RNA regulates transcription of the homeodomain transcription factors DLX5 and DLX6 in the developing mouse forebrain. We found that, in developing ventral forebrain, Evf2 recruited DLX and MECP2 transcription factors to important DNA regulatory elements in the Dlx5/6 intergenic region and controlled Dlx5, Dlx6 and Gad1 expression through trans and cis-acting mechanisms. Evf2 mouse mutants had reduced numbers of GABAergic interneurons in early postnatal hippocampus and dentate gyrus. Although the numbers of GABAergic interneurons and Gad1 RNA levels returned to normal in Evf2 mutant adult hippocampus, reduced synaptic inhibition occurred. These results suggest that noncoding RNA-dependent balanced gene regulation in embryonic brain is critical for proper formation of GABA-dependent neuronal circuitry in adult brain.

Autophosphorylation of alphaCaMKII downregulates excitability of CA1 pyramidal neurons following synaptic stimulation.

It has been well documented that alpha-calcium/calmodulin-dependent protein kinase II (alphaCaMKII) is central to synaptic plasticity such as long-term potentiation, an activity-dependent strengthening of synapses that is thought to underlie certain types of learning and memory. However, the mechanisms by which alphaCaMKII may regulate neuronal excitability remain unclear. Here, we report that alphaCaMKII knock-in mice with a targeted T286A point mutation that prevents its autophosphorylation (alphaCaMKII(T286A)) showed increased excitability of CA1 pyramidal neurons compared with wild-type controls, as measured by a decrease in the slow component of post-burst afterhyperpolarization (sAHP) following high-frequency stimulation of Schaffer collateral afferent fibers. In contrast, AHP was indistinguishable between alphaCaMKII(T286A) and wild-type mice when it was evoked by somatic current injections, indicating that the hyperexcitability is observed specifically in response to synaptic stimulation in this mutant. Taken together, our results suggest that alphaCaMKII functions to downregulate CA1 neuron excitability following synaptic stimulation, presumably supporting the functionally adaptive modulation of excitability during hippocampal learning or providing a negative feedback mechanism that would prevent neurons from becoming hyperexcitable and promote network stability.

Differential effects of alphaCaMKII mutation on hippocampal learning and changes in intrinsic neuronal excitability.

Alpha-calcium/calmodulin-dependent kinase II (alphaCaMKII) is central to synaptic plasticity but it remains unclear whether this kinase contributes to neuronal excitability changes, which are a cellular correlate of learning. Using knock-in mice with a targeted T286A mutation that prevents the autophosphorylation of alphaCaMKII (alphaCaMKII(T286A)), we studied the role of alphaCaMKII signaling in regulating hippocampal neuronal excitability during hippocampus-dependent spatial learning in the Morris water maze. Wild-type control mice showed increased excitability of CA1 pyramidal neurons, as assessed by a reduction in the postburst afterhyperpolarization (AHP), after spatial training in the water maze. Importantly, wild-type mice did not show AHP changes when they were exposed to the water maze without the escape platform and swam the same amount of time as the trained mice (swim controls), thus manifesting learning-specific increases in hippocampal CA1 excitability associated with spatial training. Meanwhile, alphaCaMKII(T286A) mice showed impairments in spatial learning but exhibited reduced levels of AHP that were similar to wild-type controls after water-maze training. Notably, both trained and swim-control groups of alphaCaMKII(T286A) mutants showed similar increased excitability, indicating that swimming by itself is enough to induce changes in excitability in the absence of normal alphaCaMKII function. This result demonstrates dissociation of alphaCaMKII-independent changes in intrinsic neuron excitability from learning and synaptic plasticity mechanisms, suggesting that increases in excitability per se are not perfectly correlated with learning. Our findings suggest that alphaCaMKII signaling may function to suppress learning-unrelated changes during training, thereby allowing hippocampal CA1 neurons to increase their excitability appropriately for encoding spatial memories.

Acute stress facilitates trace eyeblink conditioning in C57BL/6 male mice and increases the excitability of their CA1 pyramidal neurons.

The effects of stress (restraint plus tail shock) on hippocampus-dependent trace eyeblink conditioning and hippocampal excitability were examined in C57BL/6 male mice. The results indicate that the stressor significantly increased the concentration of circulating corticosterone, the amount and rate of learning relative to nonstressed conditioned mice, and the excitability of CA1 hippocampal pyramidal neurons. Behaviorally, there was no effect of the stressor on control mice that received unpaired presentations of the tone and periorbital shock, i.e., neither stressed nor nonstressed control mice showed an increase in conditioned responding that was above baseline levels. Biophysically, the stressor significantly decreased the amplitude of the post-burst afterhyperpolarization (AHP) and decreased spike frequency accommodation relative to cells from nonstressed control mice. The effect was significant for mice that were stressed either 1 h or 24 h earlier. The results suggest that the stressor increases the excitability of hippocampal pyramidal neurons and that the mechanism underlying this increase may contribute to the more rapid acquisition of hippocampally dependent eyeblink conditioning.

BACE1 deficiency rescues memory deficits and cholinergic dysfunction in a mouse model of Alzheimer's disease.

beta-site APP cleaving enzyme 1 (BACE1) is the beta-secretase enzyme required for generating pathogenic beta-amyloid (Abeta) peptides in Alzheimer's disease (AD). BACE1 knockout mice lack Abeta and are phenotypically normal, suggesting that therapeutic inhibition of BACE1 may be free of mechanism-based side effects. However, direct evidence that BACE1 inhibition would improve cognition is lacking. Here we show that BACE1 null mice engineered to overexpress human APP (BACE1(-/-).Tg2576(+)) are rescued from Abeta-dependent hippocampal memory deficits. Moreover, impaired hippocampal cholinergic regulation of neuronal excitability found in the Tg2576 AD model is ameliorated in BACE1(-/-).Tg2576(+) bigenic mice. The behavioral and electrophysiological rescue of deficits in BACE1(-/-).Tg2576(+) mice is correlated with a dramatic reduction of cerebral Abeta40 and Abeta42 levels and occurs before amyloid deposition in Tg2576 mice. Our gene-based approach demonstrates that lower Abeta levels are beneficial for AD-associated memory impairments, validating BACE1 as a therapeutic target for AD.

Watermaze learning enhances excitability of CA1 pyramidal neurons.

The dorsal hippocampus is crucial for learning the hidden-platform location in the hippocampus-dependent, spatial watermaze task. We have previously demonstrated that the postburst afterhyperpolarization (AHP) of hippocampal pyramidal neurons is reduced after acquisition of the hippocampus-dependent, temporal trace eyeblink conditioning task. We report here that the AHP and one or more of its associated currents (IAHP and/or sIAHP) are reduced in dorsal hippocampal CA1 pyramidal neurons from rats that learned the watermaze task as compared with neurons from control rats. This reduction was a learning-induced phenomenon as the AHP of CA1 neurons from rats that failed to learn the hidden-platform location was similar to that of neurons from control rats. We propose that reduction of the AHP in pyramidal neurons in regions crucial for learning is a cellular mechanism of learning that is conserved across species and tasks.

Age-related enhancement of the slow outward calcium-activated potassium current in hippocampal CA1 pyramidal neurons in vitro.

Aging is associated with learning deficits and a decrease in neuronal excitability, reflected by an enhanced post-burst afterhyperpolarization (AHP), in CA1 hippocampal pyramidal neurons. To identify the current(s) underlying the AHP altered in aging neurons, whole-cell voltage-clamp recording experiments were performed in hippocampal slices from young and aging rabbits. Similar to previous reports, aging neurons were found to rest at more hyperpolarized potentials and have larger AHPs than young neurons. Given that compounds that reduce the slow outward calcium-activated potassium current (sI(AHP)), a major constituent of the AHP, also facilitate learning in aging animals, the sI(AHP) was pharmacologically isolated and characterized. Aging neurons were found to have an enhanced sI(AHP,) the amplitude of which was significantly correlated to the amplitude of the AHP (r = 0.63; p < 0.001). Thus, an enhanced sI(AHP) contributes to the enhanced AHP in aging. No differences were found in the membrane resistance, capacitance, or kinetic and voltage-dependent properties of the sI(AHP). Because enhanced AHP in aging neurons has been hypothesized to be secondary to an enhanced Ca2+ influx via the voltage-gated L-type Ca2+ channels, we further examined the sI(AHP) in the presence of an L-type Ca2+ channel blocker, nimodipine (10 microm). Nimodipine caused quantitatively greater reductions in the sI(AHP) in aging neurons than in young neurons; however, the residual sI(AHP) was still significantly larger in aging neurons than in young neurons. Our data, in conjunction with previous studies showing a correlation between the AHP and learning, suggest that the enhancement of the sI(AHP) in aging is a mechanism that contributes to age-related learning deficits.