Noradrenergic current responses of neurons in rat oculomotor neural integrators.

The prepositus hypoglossi nucleus (PHN) and the interstitial nucleus of Cajal (INC) are involved in the control of horizontal and vertical gaze, respectively. A previous study showed that PHN neurons exhibit depolarized or hyperpolarized responses to noradrenaline (NA). However, the adrenoceptor types that participate in NA-induced responses and the effects of NA on INC neurons have not yet been investigated. Furthermore, the relationship between NA-induced responses and neuron types defined by neurotransmitter phenotypes has not been determined. In this study, we investigated NA-induced current responses in PHN and INC neurons and the relationships between these responses and neuron types using whole cell recordings in wild-type and transgenic rat brainstem slices. Local application of NA to the cell soma induced slow inward (SI) and slow outward (SO) currents that were mainly mediated by α1 and α2 adrenoceptors, respectively. These current responses were observed in both PHN and INC neurons, although the proportion of INC neurons that responded to NA was low. Analyses of the distributions of the current responses revealed that in the PHN, all fluorescently identified inhibitory neurons exhibited SI currents, whereas glutamatergic and cholinergic neurons exhibited both SI and SO currents. In the INC, glutamatergic and inhibitory neurons preferentially exhibited SI and SO currents, respectively. When the PHN and INC neurons were characterized by their firing pattern, we found that the proportions of the currents depended on their firing pattern. These results suggest that various modes of noradrenergic modulation in horizontal and vertical neural integrators are dependent on neuron type.NEW & NOTEWORTHY Noradrenergic modulation of oculomotor neural integrators involved in gaze control has not been elucidated. Here, we report that noradrenaline (NA)-induced slow inward (SI) and outward (SO) currents are mediated mainly by α1 and α2 adrenoceptors in neurons that participate in horizontal and vertical gaze control. The NA-induced current responses differed depending on the neurotransmitter phenotype and firing pattern. These results suggest various modes of noradrenergic modulation in horizontal and vertical integrator neurons.

Serotonergic current responses of neurons in rat oculomotor neural integrators.

The prepositus hypoglossi nucleus (PHN) and the interstitial nucleus of Cajal (INC) are involved in controlling horizontal and vertical gaze, respectively. Previous studies have shown that PHN neurons exhibit depolarized or hyperpolarized responses to serotonin (5-hydroxytryptamine, 5-HT). However, serotonergic modulation of INC neurons has not been examined. Furthermore, the relationship between 5-HT-induced responses and neuron types based on neurotransmitter phenotypes has not been clarified. In this study, we investigated 5-HT-induced current responses in PHN and INC neurons and the distributions of distinct current responses in different neuron types, using whole cell recordings of wild-type and transgenic rat brain stem slices. Local application of 5-HT to the cell soma confirmed that slow inward (SI) and slow outward (SO) currents were mediated by 5-HT2 and 5-HT1A receptors, respectively. Furthermore, fast inward (FI) currents that were mediated by 5-HT3 receptors were observed. These three current responses were observed in both PHN and INC neurons. Analyses of the distributions of the three current responses revealed that fluorescently identified glutamatergic and inhibitory neurons in the PHN showed high proportions of SI and SO currents, respectively, whereas glutamatergic and inhibitory neurons in the INC showed mainly SO currents. When PHN and INC neurons were characterized on the basis of firing patterns, the proportions of the currents depended on the firing patterns. The different distributions of 5-HT-induced currents suggest distinct serotonergic modulation modes specific to horizontal and vertical gaze control.NEW & NOTEWORTHY Serotonergic modulation of vertical gaze control (interstitial nucleus of Cajal, INC) is less understood than that of horizontal gaze control (prepositus hypoglossal nucleus, PHN). Here, we report 5-HT-induced fast inward currents in addition to the previously reported slow inward and outward currents. The distributions of these currents in INC neurons based on neurotransmitter phenotypes differ from those in PHN neurons. These results suggest distinct serotonergic modulation modes in horizontal and vertical gaze control centers.

Behavioral and Immunohistochemical Evidence for Suppressive Effects of Goshajinkigan on Salicylate-Induced Tinnitus in Rats.

Many people are affected by tinnitus, a sensation of ringing in the ear despite the absence of external sound. Goshajinkigan (GJG) is one of the formulations of Japanese traditional herbal medicine and is prescribed for the palliative treatment of patients with tinnitus. Although GJG is clinically effective in these patients, its behavioral effects and the underlying neuroanatomical substrate have not been modeled in animals. We modeled tinnitus using salicylate-treated rats, demonstrated the effectiveness of GJG on tinnitus, and examined the underlying neuronal substrate with c-Fos expression. Intraperitoneal injection of sodium salicylate (400 mg/kg) into rats for three consecutive days significantly increased false positive scores, which were used to assess tinnitus behavior. When GJG was orally administered one hour after each salicylate injection, the increase in tinnitus behavior was suppressed. The analysis of c-Fos expression in auditory-related brain areas revealed that GJG significantly reduced the salicylate-induced increase in the number of c-Fos-expressing cells in the auditory cortices, inferior colliculus, and dorsal cochlear nucleus. These results suggest a suppressive effect of GJG on salicylate-induced tinnitus in animal models.

Distinct purinergic receptor-mediated currents of rat oculomotor integrator neurons characterized by different firing patterns.

The prepositus hypoglossi nucleus (PHN) and the interstitial nucleus of Cajal (INC) are oculomotor neural integrators involved in the control of horizontal and vertical gaze, respectively. We previously reported that local application of adenosine 5'-trisphosphate (ATP) to PHN neurons induced P2X receptor-mediated fast inward currents, P2Y receptor-mediated slow inward currents, and/or adenosine P1 receptor-mediated slow outward currents. In contrast to the findings on PHN neurons, the expression of functional purinergic receptors in INC neurons has not been examined. In this study, we investigated ATP-induced current responses in INC neurons and the distributions of the three current types across distinct firing patterns in PHN and INC neurons using whole cell recordings of rat brainstem slices. The application of ATP induced all three current types in INC neurons. Pharmacological analyses indicated that the fast inward and slow outward currents were mainly mediated by the P2X and P1 subtypes, respectively, corresponding to the receptor subtypes in PHN neurons. However, agonists of the P2Y subtype did not induce the slow inward current in INC neurons, suggesting that other subtypes or mechanisms are responsible for this current. Analysis of the distribution of the three current types in PHN and INC neurons revealed that the proportions of the currents were distinctly dependent on the firing patterns of PHN neurons whereas the proportion of the fast inward current was higher during all firing patterns of INC neurons. The different distributions of ATP-induced currents suggest distinct modes of purinergic modulation specific to horizontal and vertical integrators.NEW & NOTEWORTHY The roles of purinergic signaling on vertical (mediated by the interstitial nucleus of Cajal; INC) and horizontal (prepositus hypoglossal nucleus; PHN) gaze control are not understood. Here, we report three current types induced by ATP in INC neurons; the distribution of these current types across different types of INC neurons is different from that in PHN neurons. These results suggest distinct modes of purinergic modulation in horizontal and vertical gaze control centers.

Distinct proportions of cholinergic neurons in the rat prepositus hypoglossi nucleus according to their cerebellar projection targets.

Cerebellar functions are modulated by cholinergic inputs, the density of which varies among cerebellar regions. Although the prepositus hypoglossi nucleus (PHN), a brainstem structure involved in controlling gaze holding, is known as one of the major sources of these cholinergic inputs, the proportions of cholinergic neurons in PHN projections to distinct cerebellar regions have not been quantitatively analyzed. In this study, we identified PHN neurons projecting to the cerebellum by applying retrograde labeling with dextran-conjugated Alexa 488 in choline acetyltransferase (ChAT)-tdTomato transgenic rats and compared the proportion of cholinergic PHN neurons in the PHN projections to four different regions of the cerebellum, namely the flocculus (FL), the uvula and nodulus (UN), lobules III-V in the vermis (VM), and the hemispheric paramedian lobule and crus 2 (PC). In the PHN, the percentage of cholinergic PHN neurons was lower in the projection to the FL than in the projection to the UN, VM or PC. Preposito-cerebellar neurons, except for preposito-FL neurons, included different proportions of cholinergic neurons at different rostrocaudal positions in the PHN. These results suggest that cholinergic PHN neurons project to not only the vestibulocerebellum but also the anterior vermis and posterior hemisphere and that the proportion of cholinergic neurons among projection neurons from the PHN differs depending on cerebellar target areas and the rostro-caudal regions of the PHN. This study provides insights regarding the involvement of cerebellar cholinergic networks in gaze holding.

Different Activation Mechanisms of Excitatory Networks in the Rat Oculomotor Integrators for Vertical and Horizontal Gaze Holding.

Gaze holding in the horizontal and vertical directions is separately controlled via the oculomotor neural integrators, the prepositus hypoglossi nucleus (PHN) and the interstitial nucleus of Cajal (INC), respectively. Our previous in vitro studies demonstrated that transient, high-frequency local stimulation of the PHN and the INC increased the frequency of spontaneous EPSCs that lasted for several seconds. The sustained EPSC response of PHN neurons was attributed to the activation of local excitatory networks primarily mediated via Ca2+-permeable AMPA (CP-AMPA) receptors and Ca2+-activated nonselective cation (CAN) channels. However, the contribution of CP-AMPA receptors to the activation of INC excitatory networks appeared to be small. In this study, we clarified the mechanisms of excitatory network activation in the PHN and INC using whole-cell recordings in rat brainstem slices. Although physiological and histological analyses showed that neurons that expressed CP-AMPA receptors existed not only in the PHN but also in the INC, the effect of a CP-AMPA receptor antagonist on the sustained EPSC response was significantly weaker in INC neurons than in PHN neurons. Meanwhile, the effect of an NMDA receptor antagonist on the sustained EPSC response was significantly stronger in INC neurons than in PHN neurons. Furthermore, the current and the charge transfer mediated via NMDA receptors were significantly larger in INC neurons than in PHN neurons. These results strongly suggest that these excitatory networks are activated via different synaptic mechanisms: a CP-AMPA receptor and CAN channel-dependent mechanism and an NMDA receptor-dependent mechanism in horizontal and vertical integrators, respectively.

Loss of HCN1 subunits causes absence epilepsy in rats.

Hyperpolarized-activated cyclic nucleotide-gated (HCN) channels underlie hyperpolarization-activated current (Ih) and are involved in controlling the excitability and electrical responsiveness of neurons. Absence epilepsy is clinically defined by a sudden, brief impairment of consciousness and behavioral arrest. Spike-and-wave discharges (SWDs) on electroencephalograms (EEG) are a diagnostic hallmark of absence epilepsy. In rat models of absence epilepsy, impaired function or expression of HCN1, a subtype of HCN channels, has been found. Here, to evaluate whether HCN1 deficiency causes absence epilepsy in rats, we developed Hcn1-knockout rats by transcription activator-like effector nuclease mutagenesis. The cortical and hippocampal pyramidal neurons of these rats displayed a significant reduction of Ih, a pronounced hyperpolarizing shift of the resting membrane potential, and increased input resistance, which indicated that the Hcn1-knockout rats were deficient in HCN1 function. The Hcn1-knockout rats were also more vulnerable to pentylenetetrazol-induced acute convulsions. More importantly, they exhibited spontaneous SWDs, which were accompanied by behavioral arrest, both of which were suppressed by ethosuximide. These results confirm the involvement of the HCN1 subunit in the regulation of input resistance and provide direct evidence that a deficiency of HCN1 caused absence epilepsy in rats.

Comparisons of Neuronal and Excitatory Network Properties between the Rat Brainstem Nuclei that Participate in Vertical and Horizontal Gaze Holding.

Gaze holding is primarily controlled by neural structures including the prepositus hypoglossi nucleus (PHN) for horizontal gaze and the interstitial nucleus of Cajal (INC) for vertical and torsional gaze. In contrast to the accumulating findings of the PHN, there is no report regarding the membrane properties of INC neurons or the local networks in the INC. In this study, to verify whether the neural structure of the INC is similar to that of the PHN, we investigated the neuronal and network properties of the INC using whole-cell recordings in rat brainstem slices. Three types of afterhyperpolarization (AHP) profiles and five firing patterns observed in PHN neurons were also observed in INC neurons. However, the overall distributions based on the AHP profile and the firing patterns of INC neurons were different from those of PHN neurons. The application of burst stimulation to a nearby site of a recorded INC neuron induced an increase in the frequency of spontaneous EPSCs. The duration of the increased EPSC frequency of INC neurons was not significantly different from that of PHN neurons. The percent of duration reduction induced by a Ca2+-permeable AMPA (CP-AMPA) receptor antagonist was significantly smaller in the INC than in the PHN. These findings suggest that local excitatory networks that activate sustained EPSC responses also exist in the INC, but their activation mechanisms including the contribution of CP-AMPA receptors differ between the INC and the PHN.

Visual experience regulates the development of long-term synaptic modifications induced by low-frequency stimulation in mouse visual cortex.

Manipulation of visual experience can considerably modify visual responses of visual cortical neurons even in adulthood in the mouse, although the modification is less profound than that observed during the critical period. Our previous studies demonstrated that low-frequency (2Hz) stimulation for 15min applied to layer 4 induces T-type Ca2+ channel-dependent long-term potentiation (LTP) at excitatory synapses in layer 2/3 neurons of visual cortex during the critical period. In this study, we investigated whether low-frequency stimulation could induce synaptic plasticity in adult mice. We found that 2Hz stimulation induced LTP of extracellular field potentials evoked by stimulation of layer 4 in layer 2/3 in adulthood as during the critical period. LTP in adulthood was blocked by L-type, but not T-type, Ca2+ channel antagonists, whereas LTP during the critical period was blocked by T-type, but not L-type, Ca2+ channel antagonists. This developmental change in LTP was prevented by dark rearing. Under pharmacological blockade of GABAA receptors, T-type Ca2+ channel-dependent LTP occurred, whereas L-type Ca2+ channel-dependent LTP did not occur. These results suggest that different forms of synaptic plasticity can contribute separately to experience-dependent modification of visual responses during the critical period and in adulthood.

Requirement of keratan sulfate proteoglycan phosphacan with a specific sulfation pattern for critical period plasticity in the visual cortex.

Proteoglycans play important roles in regulating the development and functions of the brain. They consist of a core protein and glycosaminoglycans, which are long sugar chains of repeating disaccharide units with sulfation. A recent study demonstrated that the sulfation pattern of chondroitin sulfate on proteoglycans contributes to regulation of the critical period of experience-dependent plasticity in the mouse visual cortex. In the present study, we investigated the role of keratan sulfate (KS), another glycosaminoglycan, in critical period plasticity in the mouse visual cortex. Immunohistochemical analyses demonstrated the presence of KS containing disaccharide units of N-acetylglucosamine (GlcNAc)-6-sulfate and nonsulfated galactose during the critical period, although KS containing disaccharide units of GlcNAc-6-sulfate and galactose-6-sulfate was already known to disappear before that period. The KS chains were distributed diffusely in the extracellular space and densely around the soma of a large population of excitatory and inhibitory neurons. Electron microscopic analysis revealed that the KS was localized within the perisynaptic spaces and dendrites but not in presynaptic sites. KS was mainly located on phosphacan. In mice deficient in GlcNAc-6-O-sulfotransferase 1, which is one of the enzymes necessary for the synthesis of KS chains, the expression of KS was one half that in wild-type mice. In the knockout mice, monocular deprivation during the critical period resulted in a depression of deprived-eye responses but failed to produce potentiation of nondeprived-eye responses. In addition, T-type Ca(2+) channel-dependent long-term potentiation (LTP), which occurs only during the critical period, was not observed. These results suggest that regulation by KS-phosphacan with a specific sulfation pattern is necessary for the generation of LTP and hence the potentiation of nondeprived-eye responses after monocular deprivation.

TNFα is required for the production of T-type Ca(2+) channel-dependent long-term potentiation in visual cortex.

Monocular deprivation produces depression and potentiation of visual responses evoked in visual cortical neurons by stimulation of deprived and nondeprived eyes, respectively, during the critical period of ocular dominance plasticity. Our previous studies suggested that T-type Ca(2+) channel-dependent long-term potentiation (LTP), induced by 2 Hz stimulation, mediates the potentiation of visual responses. However, it was proposed that the experience-dependent response potentiation is mediated by tumor necrosis factor-α (TNFα)-dependent homeostatic synaptic scaling but not by Hebbian synaptic plasticity, because the potentiation was absent in TNFα knockout (TNFα-KO) mice. In this study, we investigated whether TNFα is required for LTP induced by 2 Hz stimulation using visual cortical slices prepared from critical period mice and rats. The production of LTP was prevented by pharmacological blockade of TNFα in rats and mice. LTP production was also prevented by an inhibitor of TNFα-converting enzyme that converts membrane-bound TNFα to soluble TNFα. In TNFα-KO mice, LTP did not occur and was rescued by exogenous soluble TNFα. Soluble TNFα was required for LTP production only during a restricted time window soon after 2 Hz stimulation. These results strengthen the view that T-type Ca(2+) channel-dependent LTP contributes to the potentiation of nondeprived eye responses following monocular deprivation.