Hsin-wei Lu

Intrinsic and synaptic properties of vertical cells of the mouse dorsal cochlear nucleus

Multiple classes of inhibitory interneurons shape the activity of principal neurons of the dorsal cochlear nucleus (DCN), a primary target of auditory nerve fibers in the mammalian brain stem. Feedforward inhibition mediated by glycinergic vertical cells (also termed tuberculoventral or corn cells) is thought to contribute importantly to the sound-evoked response properties of principal neurons, but the cellular and synaptic properties that determine how vertical cells function are unclear. We used transgenic mice in which glycinergic neurons express green fluorescent protein (GFP) to target vertical cells for whole cell patch-clamp recordings in acute slices of DCN. We found that vertical cells express diverse intrinsic spiking properties and could fire action potentials at high, sustained spiking rates. Using paired recordings, we directly examined synapses made by vertical cells onto fusiform cells, a primary DCN principal cell type. Vertical cell synapses produced unexpectedly small-amplitude unitary currents in fusiform cells, and additional experiments indicated that multiple vertical cells must be simultaneously active to inhibit fusiform cell spike output. Paired recordings also revealed that a major source of inhibition to vertical cells comes from other vertical cells.

Physiological and morphological properties of, and effect of substance P on, neurons in the A7 catecholamine cell group in rats

The A7 catecholamine cell group consists of noradrenergic (NAergic) neurons that project to the dorsal horn of the spinal cord. Here, we characterized their morphology and physiology properties and tested the effect of substance P (Sub-P) on them, since the results of many morphological studies suggest that A7 neurons are densely innervated by Sub-P-releasing terminals from nuclei involved in the descending inhibitory system, such as the lateral hypothalamus and periaqueductal gray area. Whole cell recordings were made from neurons located approximately 200 microm rostral to the trigeminal motor nucleus (the presumed A7 area) in sagittal brainstem slices from rats aged 7-10 days. After recording, the neurons were injected with biocytin and immunostained with antibody against dopamine-beta-hydroxylase (DBH). DBH-immunoreactive (ir) cells were presumed to be NAergic neurons. They had a large somata diameter ( approximately 20 microm) and relatively simple dendritic branching patterns. They fired action potentials (AP) spontaneously with or without blockade of synaptic inputs, and had similar properties to those of NAergic neurons in other areas, including the existence of calcium channel-mediated APs and a voltage-dependent delay in initiation of the AP (an indicator of the existence of A-type potassium currents) and an ability to be hyperpolarized by norepinephrine. Furthermore, in all DBH-ir neurons tested, Sub-P caused depolarization of the membrane potential and an increase in neuronal firing rate by acting on neurokinin-1 receptors. Non-DBH-ir neurons with a smaller somata size were also found in the A7 area. These showed great diversity in firing patterns and about half were depolarized by Sub-P. Morphological examination suggested that the non-DBH-ir neurons form contacts with DBH-ir neurons. These results provide the first description of the intrinsic regulation of membrane properties of, and the excitatory effect of Sub-P on, A7 area neurons, which play an important role in pain regulation.

Spontaneous Activity Defines Effective Convergence Ratios in an Inhibitory Circuit.

Many neurons fire spontaneously, and the rate of this firing is subject to neuromodulation. How this firing affects functional connectivity within a neural network remains largely unexplored. Here we show that changes in spontaneous firing of cartwheel interneurons in the mouse dorsal cochlear nucleus (DCN) alter the effective convergence ratio of interneurons onto their postsynaptic targets through short-term synaptic plasticity. Spontaneous firing of cartwheel cells led to activity-dependent synaptic depression of individual cartwheel synapses. Depression was rapid and profound at stimulation frequencies between 10 and 200 Hz, suggesting the presence of high release probability (Pr) vesicles at these inhibitory synapses. Weak, transient synaptic facilitation could be induced after synapses were predepressed, indicating that low-Pr vesicles are also recruited, and may thus support steady-state transmission. A two-pool vesicle depletion model with 10-fold differences in Pr could account for the synaptic depression over a wide range of stimulus conditions. As a result of depression during high spontaneous activity, more cartwheel interneurons were required for effective inhibition. Convergence of four interneurons was sufficient to compensate for the effects of depression during physiologically expected rates of activity. By simulating synaptic release during spontaneous firing, we found that recruitment of low-Pr vesicles at the synapse plays a critical role in maintaining effective inhibition within a small population of interneurons. The interplay between spontaneous spiking, short-term synaptic plasticity, and vesicle recruitment thus determines the effective size of a convergent neural network. SIGNIFICANCE STATEMENT We examined the relationship between the structure of a small neural circuit and the properties of its individual synapses. Successful synaptic inhibition of a target cell firing requires a critical inhibitory synaptic strength. Synapses often become depressed during spontaneous presynaptic activity, and this increases the number of presynaptic neurons needed to mediate inhibition. We show that depression is limited by the presence of a pool of vesicles that resist depletion. Thus, the size of this vesicle pool determines the size of the circuit needed to mediate inhibition during different patterns of activity.

Neurokinin 1 receptor activates transient receptor potential-like currents in noradrenergic A7 neurons in rats.

Noradrenergic (NAergic) A7 neurons are involved in modulating nociception by releasing noradrenaline in the dorsal spinal cord. Since NAergic A7 neurons receive dense Substance P (Sub-P) releasing terminals from ventromedial medulla, here we tested the effect of Sub-P on them. Bath application of Sub-P induced an inward current (I(Sub-P)) in NAergic neurons, which was significantly blocked by Neurokinin 1 (NK1) receptor antagonist. The I(Sub-P) was reversed at approximately -20 mV, blocked by several TRP channel blockers, enhanced by OAG and negatively regulated by PKC. Immunohistochemistry staining showed that NAergic A7 neurons express high level of TRPC6 channel proteins, which is consistent with pharmacological properties of I(Sub-P) shown above, as TRPC6 channel is shown to be augmented by OAG and inhibited by PKC. In conclusion, the above results provide mechanism underlying postsynaptic action of Sub-P on NAergic A7 neurons and a role for TRPC6 channel in NAergic pain modulation.

Roles of A-type potassium currents in tuning spike frequency and integrating synaptic transmission in noradrenergic neurons of the A7 catecholamine cell group in rats

We investigated voltage-dependent K(+) currents (I(K)) in noradrenergic (NAergic) A7 neurons. The I(K) evoked consisted of A-type I(K) (I(A)), which had the characteristics of a low threshold for activation (approximately -50 mV), fast activation/inactivation, and rapid recovery from inactivation. Since the I(A) were blocked by heteropodatoxin-2 (Hptx-2), a specific Kv4 channel blocker, and the NAergic A7 neurons were shown to be reactive with antibodies against Kv4.1/Kv4.3 channel proteins, we conclude that the I(A) evoked in NAergic neurons are mediated by Kv4.1/Kv4.3 channels. I(A) were also evoked using voltage commands of a single action potential (AP), a subthreshold voltage change between two consecutive APs, or excitatory postsynaptic potential (EPSP) activity recorded in current-clamp mode (CCM). Blockade of the I(A) by 4-AP, a broad spectrum I(A) blocker, or by Hptx-2 increased the half-width and spontaneous firing of APs and reduced the amount of synaptic drive needed to elicit APs in CCM, showing that the I(A) play important roles in regulating the shape and firing frequency of APs and in synaptic integration in NAergic A7 neurons. Since these neurons are the principal projection neurons to the dorsal horn of the spinal cord, these results also suggest roles for Kv4.1/4.3 channels in descending NAergic pain regulation.

Postnatal development of noradrenergic terminals in the rat trigeminal motor nucleus: A light and electron microscopic immunocytochemical analysis.

The noradrenergic (NA) innervation in the trigeminal motor nucleus (Vmot) of postnatal and adult rats was examined by light and electron microscopic immunocytochemistry using antibodies against dopamine‐β‐hydroxylase or tyrosine hydroxylase. NA fibers were identified in the Vmot as early as the day of birth (postnatal day 0; P0). A continuous increase in the density of labeled fibers was observed during development up to P20, with a slight decrease at P30 and in the adult. Electron microscopic analysis of serial ultrathin sections revealed that, at P5, nearly half (46%) of the examined NA terminals made synaptic contact with other neuronal elements with membrane specializations. The percentage of examined NA varicosities engaged in synaptic contacts increased at P15 (74%), then decreased in the adult (64%). At all developmental ages, the majority of contacts made by these boutons were symmetrical, the postsynaptic elements being mainly dendrites and occasionally somata. Interestingly, some of the NA terminals made axo‐axon contacts with other unidentified boutons. These results show that, although the density of NA fibers increases during postnatal development, functional NA boutons are present in the Vmot at early postnatal ages. Some of these fibers might exert their effects via nonsynaptic release of noradrenaline, the so‐called volume transmission, but, in the main, they form conventional synaptic contacts with dendrites, somata, and other axonal terminals in the Vmot. These results are consistent with previous electrophysiological studies that propose an important role for the NA system in modulating mastication. Anat Rec, 290:96–107, 2007.

Slow AMPAR Synaptic Transmission Is Determined by Stargazin and Glutamate Transporters

AMPARs mediate the briefest synaptic currents in the brain by virtue of their rapid gating kinetics. However, at the mossy fiber-to-unipolar brush cell synapse in the cerebellum, AMPAR-mediated EPSCs last for hundreds of milliseconds, and it has been proposed that this time course reflects slow diffusion from a complex synaptic space. We show that upon release of glutamate, synaptic AMPARs were desensitized by transmitter by >90%. As glutamate levels subsequently fell, recovery of transmission occurred due to the presence of the AMPAR accessory protein stargazin that enhances the AMPAR response to low levels of transmitter. This gradual increase in receptor activity following desensitization accounted for the majority of synaptic transmission at this synapse. Moreover, the amplitude, duration, and shape of the synaptic response was tightly controlled by plasma membrane glutamate transporters, indicating that clearance of synaptic glutamate during the slow EPSC is dictated by an uptake process.