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The vestibular hair cell receptors of anamniotes, designated Type II, are presynaptic to bouton endings of vestibular nerve distal neurites. An additional flask-shaped hair cell receptor, Type I, is present in amniotes, and communicates with a chalice-shaped afferent neuritic ending that surrounds the entire hair cell except its apical neck. Since the full repertoire of afferent fiber dynamics and sensitivities observed throughout the vertebrate phyla can be accomplished through Type II hair cell-bouton synapses, the functional contribution(s) of Type I hair cells and their calyces to vestibular performance remains a topic of great interest. The goal of the present study was to investigate electrical coupling between the Type I hair cell and its enveloping calyx in the mouse semicircular canal crista ampullaris. Since there are no gap junctions between these two cells, evidence for electrical communication would necessarily involve other mechanisms. Simultaneous recordings from the two cells of the synaptic pair were used initially to verify the presence of orthodromic quantal synaptic transmission from the hair cell to the calyx, and then to demonstrate bi-directional communication due to the slow accumulation of potassium ions in the synaptic cleft. As a result of this potassium ion accretion, the equilibrium potentials of hair cell conductances facing the synaptic cleft become depolarized to an extent that is adequate for calcium influx into the hair cell, and the calyx inner face becomes depolarized to a level that is near the threshold for spike initiation. Following this, paired recordings were again employed to characterize fast bi-directional electrical coupling between the two cells. In this form of signaling, cleft-facing conductances in both the hair cell and calyx increase, which strengthens their coupling. Because this mechanism relies on the cleft resistance, we refer to it as resistive coupling. We conclude that the same three forms of hair cell-calyceal transmission previously demonstrated in the turtle are present in the mammalian periphery, providing a biophysical basis for the exceptional temporal fidelity of the vestibular system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Contini, Holstein and Art.)

Autophagy is a conserved, catabolic process essential for maintaining cellular homeostasis. Malfunctional autophagy contributes to neurodevelopmental and neurodegenerative diseases. However, the exact role and targets of autophagy in human neurons remain elusive. Here we report a systematic investigation of neuronal autophagy targets through integrated proteomics. Deep proteomic profiling of multiple autophagy-deficient lines of human induced neurons, mouse brains, and brain LC3-interactome reveals roles of neuronal autophagy in targeting proteins of multiple cellular organelles/pathways, including endoplasmic reticulum (ER), mitochondria, endosome, Golgi apparatus, synaptic vesicle (SV) for degradation. By combining phosphoproteomics and functional analysis in human and mouse neurons, we uncovered a function of neuronal autophagy in controlling cAMP-PKA and c-FOS-mediated neuronal activity through selective degradation of the protein kinase A - cAMP-binding regulatory (R)-subunit I (PKA-RI) complex. Lack of AKAP11 causes accumulation of the PKA-RI complex in the soma and neurites, demonstrating a constant clearance of PKA-RI complex through AKAP11-mediated degradation in neurons. Our study thus reveals the landscape of autophagy degradation in human neurons and identifies a physiological function of autophagy in controlling homeostasis of PKA-RI complex and specific PKA activity in neurons., (© 2024. The Author(s).)

In the vestibular periphery, transmission via conventional synaptic boutons is supplemented by post-synaptic calyceal endings surrounding Type I hair cells. This review focusses on the multiple modes of communication between these receptors and their enveloping calyces as revealed by simultaneous dual-electrode recordings. Classic orthodromic transmission is accompanied by two forms of bidirectional communication enabled by the extensive cleft between the Type I hair cell and its calyx. The slowest cellular communication low-pass filters the transduction current with a time constant of 10-100 ms: potassium ions accumulate in the synaptic cleft, depolarizing both the hair cell and afferent to potentials greater than necessary for rapid vesicle fusion in the receptor and potentially triggering action potentials in the afferent. On the millisecond timescale, conventional glutamatergic quantal transmission occurs when hair cells are depolarized to potentials sufficient for calcium influx and vesicle fusion. Depolarization also permits a third form of transmission that occurs over tens of microseconds, resulting from the large voltage- and ion-sensitive cleft-facing conductances in both the hair cell and the calyx that are open at their resting potentials. Current flowing out of either the hair cell or the afferent divides into the fraction flowing across the cleft into its cellular partner, and the remainder flowing out of the cleft and into the surrounding fluid compartment. These findings suggest multiple biophysical bases for the extensive repertoire of response dynamics seen in the population of primary vestibular afferent fibers. The results further suggest that evolutionary pressures drive selection for the calyx afferent., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Contini, Holstein and Art.)

A variety of stimuli activating vestibular end organs, including sinusoidal galvanic vestibular stimulation, whole body rotation and tilt, and head flexion have been shown to evoke significant changes in blood pressure (BP) and heart rate (HR). While a role for the vertical semicircular canals in altering autonomic activity has been hypothesized, studies to-date attribute the evoked BP and HR responses to the otolith organs. The present study determined whether unilateral activation of the posterior (PC) or anterior (AC) semicircular canal is sufficient to elicit changes in BP and/or HR. The study employed frequency-modulated pulsed infrared radiation (IR: 1,863 nm) directed via optical fibers to PC or AC of adult male Long-Evans rats. BP and HR changes were detected using a small-animal single pressure telemetry device implanted in the femoral artery. Eye movements evoked during IR of the vestibular endorgans were used to confirm the stimulation site. We found that sinusoidal IR delivered to either PC or AC elicited a rapid decrease in BP and HR followed by a stimulation frequency-matched modulation. The magnitude of the initial decrements in HR and BP did not correlate with the energy of the suprathreshold stimulus. This response pattern was consistent across multiple trials within an experimental session, replicable, and in most animals showed no evidence of habituation or an additive effect. Frequency modulated electrical current delivered to the PC and IR stimulation of the AC, caused decrements in HR and BP that resembled those evoked by IR of the PC. Frequency domain heart rate variability assessment revealed that, in most subjects, IR stimulation increased the low frequency (LF) component and decreased the high frequency (HF) component, resulting in an increase in the LF/HF ratio. This ratio estimates the relative contributions of sympathetic nervous system (SNS) and parasympathetic nervous system (PNS) activities. An injection of atropine, a muscarinic cholinergic receptor antagonist, diminished the IR evoked changes in HR, while the non-selective beta blocker propranolol eliminated changes in both HR and BP. This study provides direct evidence that activation of a single vertical semicircular canal is sufficient to activate and modulate central pathways that control HR and BP., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rice, Martinelli, Jiang, Holstein and Rajguru.)

Key Points: In central regions of vestibular semicircular canal epithelia, the [K + ] in the synaptic cleft ([K + ] c ) contributes to setting the hair cell and afferent membrane potentials; the potassium efflux from type I hair cells results from the interdependent gating of three conductances. Elevation of [K + ] c occurs through a calcium-activated potassium conductance, G BK , and a low-voltage-activating delayed rectifier, G K(LV) , that activates upon elevation of [K + ] c . Calcium influx that enables quantal transmission also activates I BK , an effect that can be blocked internally by BAPTA, and externally by a Ca V 1.3 antagonist or iberiotoxin. Elevation of [K + ] c or chelation of [Ca 2+ ] c linearizes the G K(LV) steady-state I-V curve, suggesting that the outward rectification observed for G K(LV) may result largely from a potassium-sensitive relief of Ca 2+ inactivation of the channel pore selectivity filter. Potassium sensitivity of hair cell and afferent conductances allows three modes of transmission: quantal, ion accumulation and resistive coupling to be multiplexed across the synapse., Abstract: In the vertebrate nervous system, ions accumulate in diffusion-limited synaptic clefts during ongoing activity. Such accumulation can be demonstrated at large appositions such as the hair cell-calyx afferent synapses present in central regions of the turtle vestibular semicircular canal epithelia. Type I hair cells influence discharge rates in their calyx afferents by modulating the potassium concentration in the synaptic cleft, [K + ] c , which regulates potassium-sensitive conductances in both hair cell and afferent. Dual recordings from synaptic pairs have demonstrated that, despite a decreased driving force due to potassium accumulation, hair cell depolarization elicits sustained outward currents in the hair cell, and a maintained inward current in the afferent. We used kinetic and pharmacological dissection of the hair cell conductances to understand the interdependence of channel gating and permeation in the context of such restricted extracellular spaces. Hair cell depolarization leads to calcium influx and activation of a large calcium-activated potassium conductance, G BK , that can be blocked by agents that disrupt calcium influx or buffer the elevation of [Ca 2+ ] i , as well as by the specific K Ca 1.1 blocker iberiotoxin. Efflux of K + through G BK can rapidly elevate [K + ] c , which speeds the activation and slows the inactivation and deactivation of a second potassium conductance, G K(LV) . Elevation of [K + ] c or chelation of [Ca 2+ ] c linearizes the G K(LV) steady-state I-V curve, consistent with a K + -dependent relief of Ca 2+ inactivation of G K(LV) . As a result, this potassium-sensitive hair cell conductance pairs with the potassium-sensitive hyperpolarization-activated cyclic nucleotide-gated channel (HCN) conductance in the afferent and creates resistive coupling at the synaptic cleft., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Anterograde and retrograde tract tracing were combined with neurotransmitter and modulator immunolabeling to identify the chemical anatomy of vestibular nuclear neurons with direct projections to the solitary nucleus in rats. Direct, sparsely branched but highly varicose axonal projections from neurons in the caudal vestibular nuclei to the solitary nucleus were observed. The vestibular neurons giving rise to these projections were predominantly located in ipsilateral medial vestibular nucleus. The cell bodies were intensely glutamate immunofluorescent, and their axonal processes contained vesicular glutamate transporter 2, supporting the interpretation that the cells utilize glutamate for neurotransmission. The glutamate-immunofluorescent, retrogradely filled vestibular cells also contained the neuromodulator imidazoleacetic acid ribotide, which is an endogenous CNS ligand that participates in blood pressure regulation. The vestibulo-solitary neurons were encapsulated by axo-somatic GABAergic terminals, suggesting that they are under tight inhibitory control. The results establish a chemoanatomical basis for transient vestibular activation of the output pathways from the caudal and intermediate regions of the solitary nucleus. In this way, changes in static head position and movement of the head in space may directly influence heart rate, blood pressure, respiration, as well as gastrointestinal motility. This would provide one anatomical explanation for the synchronous heart rate and blood pressure responses observed after peripheral vestibular activation, as well as disorders ranging from neurogenic orthostatic hypotension, postural orthostatic tachycardia syndrome, and vasovagal syncope to the nausea and vomiting associated with motion sickness. NEW & NOTEWORTHY Vestibular neurons with direct projections to the solitary nucleus utilize glutamate for neurotransmission, modulated by imidazoleacetic acid ribotide. This is the first direct demonstration of the chemical neuroanatomy of the vestibulo-solitary pathway.

Imidazole-4-acetic acid-ribotide (IAARP) is a putative neurotransmitter/modulator and an endogenous regulator of sympathetic drive, notably systemic blood pressure, through binding to imidazoline receptors. IAARP is present in neurons and processes throughout the CNS, but is particularly prevalent in regions that are involved in blood pressure control. The goal of this study was to determine whether IAARP is present in neurons in the caudal vestibular nuclei that participate in the vestibulo-sympathetic reflex (VSR) pathway. This pathway is important in modulating blood pressure upon changes in head position with regard to gravity, as occurs when humans rise from a supine position and when quadrupeds climb or rear. Sinusoidal galvanic vestibular stimulation was used to activate the VSR and cfos gene expression in VSR pathway neurons of rats. These subjects had previously received a unilateral FluoroGold tracer injection in the rostral or caudal ventrolateral medullary region. The tracer was transported retrogradely and filled vestibular neuronal somata with direct projections to the injected region. Brainstem sections through the caudal vestibular nuclei were immunostained to visualize FluoroGold, cFos protein, IAARP and glutamate immunofluorescence. The results demonstrate that IAARP is present in vestibular neurons of the VSR pathway, where it often co-localizes with intense glutamate immunofluorescence. The co-localization of IAARP and intense glutamate immunofluorescence in VSR neurons may represent an efficient chemoanatomical configuration, allowing the vestibular system to rapidly up- and down-modulate the activity of presympathetic neurons in the ventrolateral medulla, thereby altering blood pressure.

The vestibulo-sympathetic reflex (VSR) actively modulates blood pressure during changes in posture. This reflex allows humans to stand up and quadrupeds to rear or climb without a precipitous decline in cerebral perfusion. The VSR pathway conveys signals from the vestibular end organs to the caudal vestibular nuclei. These cells, in turn, project to pre-sympathetic neurons in the rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively). The present study assessed glutamate- and GABA-related immunofluorescence associated with central vestibular neurons of the VSR pathway in rats. Retrograde FluoroGold tract tracing was used to label vestibular neurons with projections to RVLM or CVLM, and sinusoidal galvanic vestibular stimulation (GVS) was employed to activate these pathways. Central vestibular neurons of the VSR were identified by co-localization of FluoroGold and cFos protein, which accumulates in some vestibular neurons following galvanic stimulation. Triple-label immunofluorescence was used to co-localize glutamate- or GABA- labeling in the identified VSR pathway neurons. Most activated projection neurons displayed intense glutamate immunofluorescence, suggestive of glutamatergic neurotransmission. To support this, anterograde tracer was injected into the caudal vestibular nuclei. Vestibular axons and terminals in RVLM and CVLM co-localized the anterograde tracer and vesicular glutamate transporter-2 signals. Other retrogradely-labeled cFos-positive neurons displayed intense GABA immunofluorescence. VSR pathway neurons of both phenotypes were present in the caudal medial and spinal vestibular nuclei, and projected to both RVLM and CVLM. As a group, however, triple-labeled vestibular cells with intense glutamate immunofluorescence were located more rostrally in the vestibular nuclei than the GABAergic neurons. Only the GABAergic VSR pathway neurons showed a target preference, projecting predominantly to CVLM. These data provide the first demonstration of two disparate chemoanatomic VSR pathways.

Protein citrullination is a calcium-driven post-translational modification proposed to play a causative role in the neurodegenerative disorders of Alzheimer's disease, multiple sclerosis (MS), and prion disease. Citrullination can result in the formation of antigenic epitopes that underlie pathogenic autoimmune responses. This phenomenon, which is best understood in rheumatoid arthritis, may play a role in the chronic dysfunction following traumatic brain injury (TBI). Despite substantial evidence of aberrations in calcium signaling following TBI, there is little understanding of how TBI alters citrullination in the brain. The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citrullination and on the specific cell types involved. Immunofluorescence revealed that controlled cortical impact in rats profoundly up--regulated protein citrullination in the cerebral cortex, external capsule, and hippocampus. This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citrullination in neurons, oligodendrocytes or microglia. Further, proteomic analyses demonstrated that the effects of TBI on citrullination were confined to a relatively small subset of neural proteins. Proteins most notably affected were those also reported to be citrullinated in other disorders, including prion disease and MS. In vivo findings were extended in an in vitro model of simulated TBI employing normal human astrocytes. Pharmacologically induced calcium excitotoxicity was shown to activate the citrullination and breakdown of glial fibrillary acidic protein, producing a novel candidate TBI biomarker and potential target for autoimmune recognition. In summary, these findings demonstrate that the effects of TBI on protein citrullination are selective with respect to brain region, cell type, and proteins modified, and may contribute to a role for autoimmune dysfunction in chronic pathology following TBI.

Spontaneous and stimulus-evoked excitatory postsynaptic currents (EPSCs) were recorded in calyx nerve terminals from the turtle vestibular lagena to quantify key attributes of quantal transmission at this synapse. On average, EPSC events had a magnitude of ∼ 42 pA, a rise time constant of τ(0) ∼ 229 μs, decayed to baseline with a time constant of τ(R) ∼ 690 μs, and carried ∼ 46 fC of charge. Individual EPSCs varied in magnitude and decay time constant. Variability in the EPSC decay time constant was hair cell dependent and due in part to a slow protraction of the EPSC in some cases. Variability in EPSC size was well described by an integer summation of unitary quanta, with each quanta of glutamate gating a unitary postsynaptic current of ∼ 23 pA. The unitary charge was ∼ 26 fC for EPSCs with a simple exponential decay and increased to ∼ 48 fC for EPSCs exhibiting a slow protraction. The EPSC magnitude and the number of simultaneous unitary quanta within each event increased with presynaptic stimulus intensity. During tonic hair cell depolarization, both the EPSC magnitude and event rate exhibited adaptive run down over time. Present data from a reptilian calyx are remarkably similar to noncalyceal vestibular synaptic terminals in diverse species, indicating that the skewed EPSC size distribution and multiquantal release might be an ancestral property of inner ear ribbon synapses., (Copyright © 2015 the American Physiological Society.)

Changes in head position and posture are detected by the vestibular system and are normally followed by rapid modifications in blood pressure. These compensatory adjustments, which allow humans to stand up without fainting, are mediated by integration of vestibular system pathways with blood pressure control centers in the ventrolateral medulla. Orthostatic hypotension can reflect altered activity of this neural circuitry. Vestibular sensory input to the vestibulo-sympathetic pathway terminates on cells in the vestibular nuclear complex, which in turn project to brainstem sites involved in the regulation of cardiovascular activity, including the rostral and caudal ventrolateral medullary regions (RVLM and CVLM, respectively). In the present study, sinusoidal galvanic vestibular stimulation was used to activate this pathway, and activated neurons were identified through detection of c-Fos protein. The retrograde tracer Fluoro-Gold was injected into the RVLM or CVLM of these animals, and immunofluorescence studies of vestibular neurons were conducted to visualize c-Fos protein and Fluoro-Gold concomitantly. We observed activated projection neurons of the vestibulo-sympathetic reflex pathway in the caudal half of the spinal, medial, and parvocellular medial vestibular nuclei. Approximately two-thirds of the cells were ipsilateral to Fluoro-Gold injection sites in both the RVLM and CVLM, and the remainder were contralateral. As a group, cells projecting to the RVLM were located slightly rostral to those with terminals in the CVLM. Individual activated projection neurons were multipolar, globular, or fusiform in shape. This study provides the first direct demonstration of the central vestibular neurons that mediate the vestibulo-sympathetic reflex., (Copyright © 2013 Wiley Periodicals, Inc.)

Present data support the conclusion that protons serve as an important neurotransmitter to convey excitatory stimuli from inner ear type I vestibular hair cells to postsynaptic calyx nerve terminals. Time-resolved pH imaging revealed stimulus-evoked extrusion of protons from hair cells and a subsequent buildup of [H(+)] within the confined chalice-shaped synaptic cleft (ΔpH ∼ -0.2). Whole-cell voltage-clamp recordings revealed a concomitant nonquantal excitatory postsynaptic current in the calyx terminal that was causally modulated by cleft acidification. The time course of [H(+)] buildup limits the speed of this intercellular signaling mechanism, but for tonic signals such as gravity, protonergic transmission offers a significant metabolic advantage over quantal excitatory postsynaptic currents--an advantage that may have driven the proliferation of postsynaptic calyx terminals in the inner ear vestibular organs of contemporary amniotes.

Sinusoidal galvanic vestibular stimulation (sGVS) induces oscillations in blood pressure (BP) and heart rate (HR), i.e., vasovagal oscillations, as well as transient decreases in BP and HR, i.e., vasovagal responses, in isoflurane-anesthetized rats. We determined the characteristics of the vasovagal oscillations, assessed their role in the generation of vasovagal responses, and determined whether they could be induced by monaural as well as by binaural sGVS and by oscillation in pitch. Wavelet analyses were used to determine the power distributions of the waveforms. Monaural and binaural sGVS and pitch generated vasovagal oscillations at the frequency and at twice the frequency of stimulation. Vasovagal oscillations and vasovagal responses were maximally induced at low stimulus frequencies (0.025-0.05 Hz). The oscillations were attenuated and the responses were rarely induced at higher stimulus frequencies. Vasovagal oscillations could occur without induction of vasovagal responses, but vasovagal responses were always associated with a vasovagal oscillation. We posit that the vasovagal oscillations originate in a low frequency band that, when appropriately activated by strong sympathetic stimulation, can generate vasovagal oscillations as a precursor for vasovagal responses and syncope. We further suggest that the activity responsible for the vasovagal oscillations arises in low frequency, otolith neurons with orientation vectors close to the vertical axis of the head. These neurons are likely to provide critical input to the vestibulo-sympathetic reflex to increase BP and HR upon changes in head position relative to gravity, and to contribute to the production of vasovagal oscillations and vasovagal responses and syncope when the baroreflex is inactivated.

Vasovagal responses (VVRs) are characterized by transient drops in blood pressure (BP) and heart rate (HR) and increased amplitude of low-frequency oscillations in the Mayer wave frequency range. Typical VVRs were induced in anesthetized, male, Long-Evans rats by sinusoidal galvanic vestibular stimulation (sGVS). VVRs were also produced by single sinusoids that transiently increased BP and HR, by 70-90° nose-up tilts, and by 60° tilts of the gravitoinertial acceleration vector using translation while rotating (TWR). The average power of the BP signal in the Mayer wave range increased substantially when tilts were >70° (0.91 g), i.e., when linear accelerations in the x-z plane were ≥0.9-1.0 g. The standard deviations of the wavelet-filtered BP signals during tilt and TWR overlaid when they were normalized to 1 g. Thus, the amplitudes of the Mayer waves coded the magnitude of the linear acceleration ≥1 g acting on the head and body, and the average power in this frequency range was associated with the generation of VVRs. These data show that VVRs are a natural outcome of stimulation of the vestibulosympathetic reflex and are not a disease. The results also demonstrate the usefulness of the rat as a small animal model for studying human VVRs.

This article reviews the structure function of the vestibular system and its pathology with respect to requirements for the design and construction of a functional vestibular prosthesis. The ultimate goal of a vestibular prosthesis is to restore balance and equilibrium through direct activation of vestibular nerve fibers. An overview of the peripheral and central vestibular systems that highlights their most important functional aspects re: the design of a prosthesis is provided. Namely, the peripheral labyrinth faithfully transduces head motion and gravity in both the time and frequency domains. These signals are described in hopes that they may be prosthetically replicated. The peripheral and central connections of the vestibular nerve are also discussed in detail, as are the vestibular nuclei in the brainstem that receive VIIIth nerve innervation. Lastly, the functional effector pathways of the vestibular system, including the vestibulo-ocular, vestibulo-spinal, vestibulo-colic, vestibulo-autonomic, and vestibular efferent innervation of the labyrinth are reviewed., (Copyright © 2012 Wiley Periodicals, Inc.)

Parkinson's disease (PD) is characterized pathologically by the formation of ubiquitin and α-synuclein (α-syn)-containing inclusions (Lewy bodies), dystrophic dopamine (DA) terminals, and degeneration of midbrain DA neurons. The precise molecular mechanisms underlying these pathological features remain elusive. Accumulating evidence has implicated dysfunctional autophagy, the cell self-digestion and neuroprotective pathway, as one of the pathogenic systems contributing to the development of idiopathic PD. Here we characterize autophagy-deficient mouse models and provide in vivo evidence for the potential role that impaired autophagy plays in pathogenesis associated with PD. Cell-specific deletion of essential autophagy gene Atg7 in midbrain DA neurons causes delayed neurodegeneration, accompanied by late-onset locomotor deficits. In contrast, Atg7-deficient DA neurons in the midbrain exhibit early dendritic and axonal dystrophy, reduced striatal dopamine content, and the formation of somatic and dendritic ubiquitinated inclusions in DA neurons. Furthermore, whole-brain-specific loss of Atg7 leads to presynaptic accumulation of α-syn and LRRK2 proteins, which are encoded by two autosomal dominantly inherited PD-related genes. Our results suggest that disrupted autophagy may be associated with enhanced levels of endogenous α-syn and LRRK2 proteins in vivo. Our findings implicate dysfunctional autophagy as one of the failing cellular mechanisms involved in the pathogenesis of idiopathic PD.

The vestibular system sends projections to brainstem autonomic nuclei that modulate heart rate and blood pressure in response to changes in head and body position with regard to gravity. Consistent with this, binaural sinusoidally modulated galvanic vestibular stimulation (sGVS) in humans causes vasoconstriction in the legs, while low frequency (0.02-0.04 Hz) sGVS causes a rapid drop in heart rate and blood pressure in anesthetized rats. We have hypothesized that these responses occur through activation of vestibulo-sympathetic pathways. In the present study, c-Fos protein expression was examined in neurons of the vestibular nuclei and rostral ventrolateral medullary region (RVLM) that were activated by low frequency sGVS. We found c-Fos-labeled neurons in the spinal, medial, and superior vestibular nuclei (SpVN, MVN, and SVN, respectively) and the parasolitary nucleus. The highest density of c-Fos-positive vestibular nuclear neurons was observed in MVN, where immunolabeled cells were present throughout the rostro-caudal extent of the nucleus. c-Fos expression was concentrated in the parvocellular region and largely absent from magnocellular MVN. c-Fos-labeled cells were scattered throughout caudal SpVN, and the immunostained neurons in SVN were restricted to a discrete wedge-shaped area immediately lateral to the IVth ventricle. Immunofluorescence localization of c-Fos and glutamate revealed that approximately one third of the c-Fos-labeled vestibular neurons showed intense glutamate-like immunofluorescence, far in excess of the stain reflecting the metabolic pool of cytoplasmic glutamate. In the RVLM, which receives a direct projection from the vestibular nuclei and sends efferents to preganglionic sympathetic neurons in the spinal cord, we observed an approximately threefold increase in c-Fos labeling in the sGVS-activated rats. We conclude that localization of c-Fos protein following sGVS is a reliable marker for sGVS-activated neurons of the vestibulo-sympathetic pathway.

We have previously demonstrated that imidazole-4-acetic acid-ribotide (IAA-RP) is present in the mammalian brain and is an endogenous ligand at imidazoline binding sites. In the present study, we used a polyclonal antiserum to visualize IAA-RP-containing neurons in the rat caudoputamen. We observe IAA-RP-immunostained neurons scattered throughout the dorsal and ventral striatum. Most of these cells co-localize GABA, but none are parvalbumin-immunoreactive. In contrast, approximately 50% of the calbindin D28k-immunopositive striatal neurons co-localize IAA-RP. Electrophysiological studies using corticostriatal slices demonstrated that bath application of IAA-RP reversibly depresses the synaptically mediated component of field potentials recorded in the striatum by stimulation of cortical axons. Addition of competitive glutamate receptor antagonists completely blocks the response, confirming its association with glutamatergic transmission. Using paired-pulse stimuli, IAA-RP was shown to exert, at least in part, a presynaptic effect, but blockade of GABAA receptor-mediated transmission did not alter the response. Lastly, we show that this effect is attributable to imidazoline-1 receptors, and not to α2 adrenergic receptors. Since IAA-RP is an endogenous central regulator of blood pressure, and cardiovascular dysfunction is a common symptom associated with Parkinson's disease (PD), we speculate that IAA-RP-related abnormalities may underlie some of the autonomic dysfunction that occurs in PD.

Blood pressure (BP) and heart rate (HR) were studied in isoflurane-anesthetized Long-Evans rats during sinusoidal galvanic vestibular stimulation (sGVS) and sinusoidal oscillation in pitch to characterize vestibular influences on autonomic control of BP and HR. sGVS was delivered binaurally via Ag/AgCl needle electrodes inserted over the mastoids at stimulus frequencies 0.008-0.4 Hz. Two processes affecting BP and HR were induced by sGVS: 1) a transient drop in BP (≈15-20 mmHg) and HR (≈3 beat*s(-1)), followed by a slow recovery over 1-6 min; and 2) inhibitory modulations in BP (≈4.5 mmHg/g) and HR (≈0.15 beats*s(-1)/g) twice in each stimulus cycle. The BP and HR modulations were approximately in-phase with each other and were best evoked by low stimulus frequencies. A wavelet analysis indicated significant energies in BP and HR at scales related to twice and four times the stimulus frequency bands. BP and HR were also modulated by oscillation in pitch at frequencies 0.025-0.5 Hz. Sensitivities at 0.025 Hz were ≈4.5 mmHg/g (BP) and ≈0.17 beat*s(-1)/g (HR) for pitches of 20-90°. The tilt-induced BP and HR modulations were out-of-phase, but the frequencies at which responses were elicited by tilt and sGVS were the same. The results show that the sGVS-induced responses, which likely originate in the otolith organs, can exert a powerful inhibitory effect on both BP and HR at low frequencies. These responses have a striking resemblance to human vasovagal responses. Thus, sGVS-activated rats can potentially serve as a useful experimental model of the vasovagal response in humans.

The present results show that the semicircular canal crista ampullaris of the toadfish, Opsanus tau, is sensitive to infrared radiation (IR) applied in vivo. IR pulse trains (∼1862 nm, ∼200 μs pulse⁻¹) delivered to the sensory epithelium by an optical fibre evoked profound changes in phasic and tonic discharge rates of postsynaptic afferent neurons. Phasic afferent responses to pulsed IR occurred with a latency of <8 ms while tonic responses developed with a time constant (τ) of 7 ms to 10 s following the onset or cessation of the radiation. Afferents responded to direct optical radiation of the sensory epithelium but did not respond to thermal stimuli that generated nearly equivalent temperature increases of the whole organ. A subset of afferent neurons fired an action potential in response to each IR pulse delivered to the sensory epithelium, at phase-locked rates up to 96 pulses per second. The latency between IR pulses and afferent nerve action potentials was much greater than synaptic delay and spike generation, demonstrating the presence of a signalling delay interposed between the IR pulse and the action potential. The same IR stimulus applied to afferent nerve axons failed to evoke responses of similar magnitude and failed to phase-lock afferent nerve action potentials. The present data support the hypothesis that pulsed IR activates sensory hair cells, thus leading to modulation of synaptic transmission and afferent nerve discharge reported here.

Imidazole-4-acetic acid-ribotide (IAA-RP), an endogenous agonist at imidazoline receptors (I-Rs), is a putative neurotransmitter/regulator in mammalian brain. We studied the effects of IAA-RP on excitatory transmission by performing extracellular and whole cell recordings at Schaffer collateral-CA1 synapses in rat hippocampal slices. Bath-applied IAA-RP induced a concentration-dependent depression of synaptic transmission that, after washout, returned to baseline within 20 min. Maximal decrease occurred with 10 μM IAA-RP, which reduced the slope of field extracellular postsynaptic potentials (fEPSPs) to 51.2 ± 5.7% of baseline at 20 min of exposure. Imidazole-4-acetic acid-riboside (IAA-R; 10 μM), the endogenous dephosphorylated metabolite of IAA-RP, also produced inhibition of fEPSPs. This effect was smaller than that produced by IAA-RP (to 65.9 ± 3.8% of baseline) and occurred after a further 5- to 8-min delay. The frequency, but not the amplitude, of miniature excitatory postsynaptic currents was decreased, and paired-pulse facilitation (PPF) was increased after application of IAA-RP, suggesting a principally presynaptic site of action. Since IAA-RP also has low affinity for α(2)-adrenergic receptors (α(2)-ARs), we tested synaptic depression induced by IAA-RP in the presence of α(2)-ARs, I(1)-R, or I(3)-R antagonists. The α(2)-AR antagonist rauwolscine (100 nM), which blocked the actions of the α(2)-AR agonist clonidine, did not affect either the IAA-RP-induced synaptic depression or the increase in PPF. In contrast, efaroxan (50 μM), a mixed I(1)-R and α(2)-AR antagonist, abolished the synaptic depression induced by IAA-RP and abolished the related increase in PPF. KU-14R, an I(3)-R antagonist, partially attenuated responses to IAA-RP. Taken together, these data support a role for IAA-RP in modulating synaptic transmission in the hippocampus through activation of I-Rs.

While the basic pathways mediating vestibulo-ocular, -spinal, and -collic reflexes have been described in detail, little is known about vestibular projections to central autonomic sites. Previous studies have primarily focused on projections from the caudal vestibular region to solitary, vagal and parabrachial nuclei, but have noted a sparse innervation of the ventrolateral medulla. Since a direct pathway from the vestibular nuclei to the rostral ventrolateral medulla would provide a morphological substrate for rapid modifications in blood pressure, heart rate and respiration with changes in posture and locomotion, the present study examined anatomical evidence for this pathway using anterograde and retrograde tract tracing and immunofluorescence detection in brainstem sections of the rat medulla. The results provide anatomical evidence for direct pathways from the caudal vestibular nuclear complex to the rostral and caudal ventrolateral medullary regions. The projections are conveyed by fine and highly varicose axons that ramify bilaterally, with greater terminal densities present ipsilateral to the injection site and more rostrally in the ventrolateral medulla. In the rostral ventrolateral medulla, these processes are highly branched and extremely varicose, primarily directed toward the somata and proximal dendrites of non-catecholaminergic neurons, with minor projections to the distal dendrites of catecholaminergic cells. In the caudal ventrolateral medulla, the axons of vestibular nucleus neurons are more modestly branched with fewer varicosities, and their endings are contiguous with both the perikarya and dendrites of catecholamine-containing neurons. These data suggest that vestibular neurons preferentially target the rostral ventrolateral medulla, and can thereby provide a morphological basis for a short latency vestibulo-sympathetic pathway., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

The vestibular system senses the movement and position of the head in space and uses this information to stabilize vision, control posture, perceive head orientation and self-motion in three-dimensional space, and modulate autonomic and limbic activity in response to locomotion and changes in posture. Most vestibular signals are not consciously perceived and are usually appreciated through effector pathways classically described as the vestibulo-ocular, vestibulo-spinal, vestibulo-collic and vestibulo-autonomic reflexes. The present study reviews some of the recent data concerning the connectivity and chemical anatomy of vestibular projections to autonomic sites that are important in the sympathetic control of blood pressure.

We followed the development of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) system during locust embryogenesis in whole mount nervous systems and brain sections by using various cytochemical techniques. We visualized NO-sensitive neurons by cGMP immunofluorescence after incubation with an NO donor in the presence of the soluble guanylyl cyclase (sGC) activator YC-1 and the phosphodiesterase-inhibitor isobutyl-methyl-xanthine (IBMX). Central nervous system (CNS) cells respond to NO as early as 38% embryogenesis. By using the NADPH-diaphorase technique, we identified somata and neurites of possible NO-synthesizing cells in the CNS. The first NADPH-diaphorase-positive cell bodies appear around 40% embryogenesis in the brain and at 47% in the ventral nerve cord. The number of positive cells reaches the full complement of adult cells at 80%. In the brain, some structures, e.g., the mushroom bodies acquire NADPH-diaphorase staining only postembryonically. Immunolocalization of L-citrulline confirmed the presence of NOS in NADPH-diaphorase-stained neurons and, in addition, indicated enzymatic activity in vivo. In whole mount ventral nerve cords, citrulline immunolabeling was present in varying subsets of NADPH-diaphorase-positive cells, but staining was very variable and often weak. However, in a regeneration paradigm in which one of the two connectives between ganglia had been crushed, strong, reliable staining was observed as early as 60% embryogenesis. Thus, citrulline immunolabeling appears to reflect specific activity of NOS. However, in younger embryos, NOS may not always be constitutively active or may be so at a very low level, below the citrulline antibody detection threshold. For the CNS, histochemical markers for NOS do not provide conclusive evidence for a developmental role of this enzyme., ((c) 2009 Wiley-Liss, Inc.)

Binaural computations involving the convergence of excitatory and inhibitory inputs have been proposed to explain directional sharpening and frequency tuning documented in the brainstem of a teleost fish, the oyster toadfish (Opsanus tau). To assess the presence of inhibitory neurons in the ascending auditory circuit, we used a monoclonal antibody to GABA to evaluate immunoreactivity at three levels of the circuit: the first order descending octaval nucleus (DON), the secondary octaval population (dorsal division), and the midbrain torus semicircularis. We observed a subset of immunoreactive (IR) cells and puncta distributed throughout the neuropil at all three locations. To assess whether contralateral inhibition is present, fluorescent dextran crystals were inserted into dorsal DON to fill contralateral, commissural inputs retrogradely prior to GABA immunohistochemistry. GABA-IR somata and puncta co-occurred with retrogradely filled, GABA-negative auditory projection cells. GABA-IR projection cells were more common in the dorsolateral DON than in the dorsomedial DON, but GABA-IR puncta were common in both dorsolateral and dorsomedial divisions. Our findings demonstrate that GABA is present in the ascending auditory circuit in the brainstem of the toadfish, indicating that GABA-mediated inhibition participates in shaping auditory response characteristics in a teleost fish as in other vertebrates., (Copyright 2010 Elsevier B.V. All rights reserved.)

Nitric oxide (NO) is a gaseous messenger molecule formed during conversion of L: -arginine into L: -citrulline by the enzyme NO synthase (NOS), which belongs to a group of NADPH diaphorases. Because of its gaseous diffusion properties, NO differs from classical neurotransmitters in that it is not restricted to synaptic terminals. In target cells, NO activates soluble guanylyl cyclase leading to an increase in cGMP levels. In insects, this NO/cGMP-signalling pathway is involved in development, memory formation and processing of visual, olfactory and mechanosensory information. We have analysed the distribution of putative NO donor and target cells in the central complex, a brain area involved in sky-compass orientation, of the locust Schistocerca gregaria by immunostaining for L: -citrulline and cGMP. Six types of citrulline-immunostained neurons have been identified including a bilateral pair of hitherto undescribed neurons that connect the lateral accessory lobes with areas anterior to the medial lobes of the mushroom bodies. Three-dimensional reconstructions have revealed the connectivity pattern of a set of 18 immunostained pontine neurons of the central body. All these neurons appear to be a subset of previously mapped NADPH-diaphorase-positive neurons of the central complex. At least three types of central-complex neurons show cGMP immunostaining including a system of novel columnar neurons connecting the upper division of the central body and the lateral triangle of the lateral accessory lobe. Our results provide the morphological basis for further studies of the function of the labelled neurons and new insights into NO/cGMP signalling.

Despite pharmacological and immunohistochemical evidence for GABA as a neurotransmitter in the olivocochlear efferent bundle, a clear functional role of GABA in the inner ear has not emerged. To explore the role of metabotropic GABA(B) receptors, we characterized the cochlear phenotype of mice with targeted deletion of the GABA(B1) subunit and determined its tissue localization using a mouse line expressing a GFP-tagged GABA(B1) subunit under the endogenous promoter. Immunostaining revealed GABA(B1) expression in both type I and type II ganglion cells and in their synaptic terminals under inner and outer hair cells, respectively. No GABA(B1) expression was observed in hair cells. Mean cochlear thresholds, measured via both auditory brainstem responses and distortion product otoacoustic emissions (DPOAEs), were elevated by approximately 10 dB in GABA(B1)-deficient mice, consistent with outer hair cell dysfunction. Olivocochlear efferent function, assessed via DPOAE suppression during efferent electrical stimulation, was unaffected by GABA(B1) deletion. GABA(B1)-deficient mice showed increased resistance to permanent acoustic injury, with mean threshold shifts approximately 25 dB smaller than wild-types after exposure to 8-16-kHz noise at 100 dB for 2 h. In contrast, there was no vulnerability difference to temporary acoustic injury following exposure to the same noise at 94 dB for 15 min. Our results suggest that GABAergic signaling in type II afferent neurons may be required for normal outer hair cell amplifier function at low sound levels and may also modulate outer hair cell responses to high-level sound.

Autophagy is a cellular self-eating process that plays an important role in neuroprotection as well as neuronal injury and death. The detailed pathway of autophagy in these two opposing functions remains to be elucidated. Neurons are highly specialized, postmitotic cells that are typically composed of a soma (cell body), a dendritic tree, and an axon. Here, we describe methods for studying autophagy in the central nervous system (CNS). The first involves the use of recently developed transgenic mice expressing the fluorescent autophagosome marker, GFP-LC3. Although CNS neurons show little evidence for the presence of GFP-LC3-containing puncta under normal conditions, under pathological conditions such neurons exhibit many GFP-LC3 puncta. The onset and density of GFP-LC3 puncta have been found to vary significantly in the subcompartments of the affected neurons. These studies suggest that autophagy is distinctly regulated in CNS neurons and that neuronal autophagy can be highly compartmentalized. While transgenic mice expressing GFP-LC3 are a valuable tool for assessing autophagic activity in the CNS, caution needs to be taken when interpreting results solely based on the presence of GFP-LC3 puncta. Therefore, traditional ultrastructural analysis using electron microscopy remains an important tool for studying autophagosomes in vivo. Additional reporters of autophagy are constantly being sought. For example, recently a selective substrate of autophagy p62/SQSTM1 has been shown to be specifically regulated by autophagic activity. Therefore, p62/SQSTM1 protein levels can be used as an additional reporter for autophagic activity.

The central complex of acridid grasshoppers integrates sensory information pertinent to reproduction-related acoustic communication. Activation of nitric oxide (NO)/cyclic GMP-signaling by injection of NO donors into the central complex of restrained Chorthippus biguttulus females suppresses muscarine-stimulated sound production. In contrast, sound production is released by aminoguanidine (AG)-mediated inhibition of nitric oxide synthase (NOS) in the central body, suggesting a basal release of NO that suppresses singing in this situation. Using anti-citrulline immunocytochemistry to detect recent NO production, subtypes of columnar neurons with somata located in the pars intercerebralis and tangential neurons with somata in the ventro-median protocerebrum were distinctly labeled. Their arborizations in the central body upper division overlap with expression patterns for NOS and with the site of injection where NO donors suppress sound production. Systemic application of AG increases the responsiveness of unrestrained females to male calling songs. Identical treatment with the NOS inhibitor that increased male song-stimulated sound production in females induced a marked reduction of citrulline accumulation in central complex columnar and tangential neurons. We conclude that behavioral situations that are unfavorable for sound production (like being restrained) activate NOS-expressing central body neurons to release NO and elevate the behavioral threshold for sound production in female grasshoppers.

The development and cellular distribution of the inhibitory neurotransmitter glycine in the spinal cord of the sea lamprey were studied by immunocytochemistry and double immunofluorescence and compared with the distribution of gamma-aminobutyric acid (GABA). Results in lamprey embryos and prolarvae reveal that the appearance of glycine-immunoreactive (-ir) spinal neurons precedes that of GABA-ir neurons. Throughout development, glycine-ir cells in the lateral and dorsomedial gray matter of the spinal cord are more numerous than the GABA-ir cells. Only a subset of these neurons shows colocalization of GABA and glycine, suggesting that they are primarily disparate neuronal populations. In contrast, most cerebrospinal fluid (CSF)-contacting neurons of the central canal walls are strongly GABA-ir, and only a portion of them are faintly glycine-ir. Some edge cells (lamprey intraspinal mechanoreceptors) were glycine-ir in larvae and adults. The glycine-ir and GABA-ir neuronal populations observed in the adult spinal cord were similar to those found in larvae. Comparison of glycine-ir and GABA-ir fibers coursing longitudinally in the spinal cord of adult lamprey revealed large differences in diameter between these two types of fiber. Commissural glycine-ir fibers appear in prolarvae and become numerous at larval stages, whereas crossed GABA-ir are scarce. Taken together, results in this primitive vertebrate indicate that the spinal glycinergic cells do not arise by biochemical shift of preexisting GABAergic cells but instead suggest that glycine is present in the earliest circuitry of the developing lamprey spinal cord, where it might act transiently as an excitatory transmitter., ((c) 2008 Wiley-Liss, Inc.)

Autophagy is a regulated lysosomal degradation process that involves autophagosome formation and transport. Although recent evidence indicates that basal levels of autophagy protect against neurodegeneration, the exact mechanism whereby this occurs is not known. By using conditional knockout mutant mice, we report that neuronal autophagy is particularly important for the maintenance of local homeostasis of axon terminals and protection against axonal degeneration. We show that specific ablation of an essential autophagy gene, Atg7, in Purkinje cells initially causes cell-autonomous, progressive dystrophy (manifested by axonal swellings) and degeneration of the axon terminals. Consistent with suppression of autophagy, no autophagosomes are observed in these dystrophic swellings, which is in contrast to accumulation of autophagosomes in the axonal dystrophic swellings under pathological conditions. Axonal dystrophy of mutant Purkinje cells proceeds with little sign of dendritic or spine atrophy, indicating that axon terminals are much more vulnerable to autophagy impairment than dendrites. This early pathological event in the axons is followed by cell-autonomous Purkinje cell death and mouse behavioral deficits. Furthermore, ultrastructural analyses of mutant Purkinje cells reveal an accumulation of aberrant membrane structures in the axonal dystrophic swellings. Finally, we observe double-membrane vacuole-like structures in wild-type Purkinje cell axons, whereas these structures are abolished in mutant Purkinje cell axons. Thus, we conclude that the autophagy protein Atg7 is required for membrane trafficking and turnover in the axons. Our study implicates impairment of axonal autophagy as a possible mechanism for axonopathy associated with neurodegeneration.

Considerable research has been devoted to the understanding of how nitric oxide (NO) influences brain function. Few studies, however, have addressed how its production is physiologically regulated. Here, we report that protein-protein interactions between neuronal NO synthase (nNOS) and glutamate NMDA receptors via the scaffolding protein postsynaptic density-95 (PSD-95) in the hypothalamic preoptic region of adult female rats is sensitive to cyclic estrogen fluctuation. Coimmunoprecipitation experiments were used to assess the physical association between nNOS and NMDA receptor NR2B subunit in the preoptic region of the hypothalamus. We found that nNOS strongly interacts with NR2B at the onset of the preovulatory surge at proestrus (when estrogen levels are highest) compared with basal-stage diestrous rats. Consistently, estrogen treatment of gonadectomized female rats also increases nNOS/NR2B complex formation. Moreover, endogenous fluctuations in estrogen levels during the estrous cycle coincide with changes in the physical association of nNOS to PSD-95 and the magnitude of NO release in the preoptic region. Finally, temporary and local in vivo suppression of PSD-95 synthesis by using antisense oligodeoxynucleotides leads to inhibition of nNOS activity in the preoptic region and disrupted estrous cyclicity, a process requiring coordinated activation of neurons containing gonadotropin-releasing hormone (the neuropeptide controlling reproductive function). In conclusion, our findings identify a novel steroid-mediated molecular mechanism that enables the adult mammalian brain to control NO release under physiological conditions.

A substantial body of research has led to the recognition that the vestibular system participates in blood pressure modulation during active movements and changes in posture, and that this modulation is effected at least partly by the caudal vestibular nuclei. The I-4 isomer of imidazoleacetic acid-ribotide (IAA-RP) is a putative neurotransmitter/modulator that is thought to be an endogenous regulator of general sympathetic drive, particularly systemic blood pressure. The present study employed immunofluorescence and light and electron microscopic immunocytochemistry to visualize IAA-RP in the vestibular nuclei of adult male rats. The results demonstrate IAA-RP immunolabeling of subpopulations of vestibular neurons in the descending nucleus and the caudal half of the medial nucleus, with scattered immunostained vestibular neurons also present more rostrally. On the basis of double immunofluorescence staining for IAA-RP and calbindin, many of these ribotide-immunoreactive neurons appear to be innervated by cerebellar Purkinje cell afferents. Ultrastructural observations in the caudal vestibular nuclei confirm the IAA-RP immunolocalization in cell bodies and dendritic processes, and in some myelinated axons and presynaptic boutons. The regional distribution of IAA-RP immunoreactivity corresponds to the location of vestibular neurons involved in autonomic functions. The presence of IAA-RP in those neurons suggests that they participate specifically in vestibulo-autonomic regulation of blood pressure. The localization of immunostain in processes and terminals suggests that vestibulo-autonomic activity is subject to local feedback control. Overall, the observations offer a chemoanatomic basis for understanding the vestibular side effects commonly experienced by patients treated with clonidine and other imidazoline-related drugs., ((c) 2007 Wiley-Liss, Inc.)

Imidazoleacetic acid-ribotide (IAA-RP) is a putative neurotransmitter/modulator recently discovered in mammalian brain. The present study examines the distribution of IAA-RP in the rat CNS using a highly specific antiserum raised in rabbit against IAA-RP with immunostaining of aldehyde-fixed rat CNS. IAA-RP-immunoreactive neurons were present throughout the neuraxis; neuroglia were not labeled. In each region, only a subset of the neuronal pool was immunostained. In the forebrain, ribotide-immunolabeled neurons were common in neocortex, in hippocampal formation, and in subcortical structures including basal ganglia, thalamus and hypothalamus. Labeling was prominent in limbic areas including olfactory bulb, basal forebrain, pyriform cortex and amygdala. In the mid- and hindbrain, immunolabeled neurons were concentrated in specific nuclei and, in some areas, in specific subregions of those nuclei. Structures of the motor system, including cranial nerve motor nuclei, precerebellar nuclei, the substantia nigra, and the red nucleus were clearly labeled. Staining was intense in cells and/or puncta in the rostral and caudal ventrolateral medullary reticular formation, nucleus tractus solitarius and the caudal vestibular nuclear complex. Within neurons, the ribotide was found predominantly in somata and dendrites; some myelinated axons and occasional synaptic terminals were also immunostained. These data indicate that IAA-RP contributes to the neurochemical phenotype of many neuronal populations and further supports our suggestion that, in autonomic structures, IAA-RP may serve as a chemical mediator in complex circuits involved in blood pressure regulation and, more generally, sympathetic drive.

The neurochemistry of the retina of the larval and postmetamorphic sea lamprey was studied via immunocytochemistry using antibodies directed against the major candidate neurotransmitters [glutamate, glycine, gamma-aminobutyric acid (GABA), aspartate, dopamine, serotonin] and the neurotransmitter-synthesizing enzyme tyrosine hydroxylase. Immunoreactivity to rod opsin and calretinin was also used to distinguish some retinal cells. Two retinal regions are present in larvae: the central retina, with opsin-immunoreactive photoreceptors, and the lateral retina, which lacks photoreceptors and is mainly neuroblastic. We observed calretinin-immunostained ganglion cells in both retinal regions; immunolabeled bipolar cells were detected in the central retina only. Glutamate immunoreactivity was present in photoreceptors, ganglion cells, and bipolar cells. Faint to moderate glycine immunostaining was observed in photoreceptors and some cells of the ganglion cell/inner plexiform layer. No GABA-immunolabeled perikarya were observed. GABA-immunoreactive centrifugal fibers were present in the central and lateral retina. These centrifugal fibers contacted glutamate-immunostained ganglion cells. No aspartate, serotonin, dopamine, or TH immunoreactivity was observed in larvae, whereas these molecules, as well as GABA, glycine, and glutamate, were detected in neurons of the retina of recently transformed lamprey. Immunoreactivity to GABA was observed in outer horizontal cells, some bipolar cells, and numerous amacrine cells, whereas immunoreactivity to glycine was found in amacrine cells and interplexiform cells. Dopamine and serotonin immunoreactivity was found in scattered amacrine cells. Amacrine and horizontal cells did not express classical neurotransmitters (with the possible exception of glycine) during larval life, so transmitter-expressing cells of the larval retina appear to participate only in the vertical processing pathway.

The present immunocytochemical study provides evidence of a previously unrecognized, rich, gamma-aminobutyric acid (GABA)-ergic innervation of the pineal organ in the dogfish (Scyliorhinus canicula). In this elasmobranch, the pineal primordium is initially detected at embryonic stage 24 and grows to form a long pineal tube by stage 28. Glutamic acid decarboxylase (GAD)-immunoreactive (-ir) fibers were first observed at stage 26, and by stage 28, thin GAD-ir fibers were detectable at the base of the pineal neuroepithelium. In pre-hatchling embryos, most fibers gave rise to GAD-ir boutons that were localized in the basal region of the neuroepithelium, although a smaller number of labeled terminals ascended to the pineal lumen. A few pale GAD-ir perikarya were observed within the pineal organ of stage 29 embryos, but GAD-ir perikarya were not observed at other developing stages or in adults. In contrast, GABA immunocytochemistry revealed the presence of GABAergic perikarya and fibers in the pineal organ of late stage embryos and adults. Although high densities of GABAergic cells were observed in the paracommissural pretectum, posterior tubercle, and tegmentum of dogfish embryos (regions previously demonstrated to contain pinealopetal cells), the presence of GABA-ir perikarya in the pineal organ strongly suggests that the rich GABAergic innervation of the elasmobranch pineal organ is intrinsic. This contrasts with the central origin of GABAergic fibers in the pineal gland of some mammals.

The vestibular portion of the eighth cranial nerve informs the brain about the linear and angular movements of the head in space and the position of the head with respect to gravity. The termination sites of these eighth nerve afferents define the territory of the vestibular nuclei in the brainstem. (There is also a subset of afferents that project directly to the cerebellum.) This chapter reviews the anatomical organization of the vestibular nuclei, and the anatomy of the pathways from the nuclei to various target areas in the brain. The cytoarchitectonics of the vestibular brainstem are discussed, since these features have been used to distinguish the individual nuclei. The neurochemical phenotype of vestibular neurons and pathways are also summarized because the chemical anatomy of the system contributes to its signal-processing capabilities. Similarly, the morphologic features of short-axon local circuit neurons and long-axon cells with extrinsic projections are described in detail, since these structural attributes of the neurons are critical to their functional potential. Finally, the composition and hodology of the afferent and efferent pathways of the vestibular nuclei are discussed. In sum, this chapter reviews the morphology, chemoanatomy, connectivity, and synaptology of the vestibular nuclei.

In the adult cricket, neurogenesis occurs in the mushroom bodies, the main integrative structures of the insect brain. Mushroom body neuroblast proliferation is modulated in response to environmental stimuli. However, the mechanisms underlying these effects remain unspecified. In the present study, we demonstrate that electrical stimulation of the antennal nerve mimics the effects of olfactory activation and increases mushroom body neurogenesis. The putative role of nitric oxide (NO) in this activity-regulated neurogenesis was then explored. In vivo and in vitro experiments demonstrate that NO synthase inhibition decreases, and NO donor application stimulates neuroblast proliferation. NADPH-d activity, anti-L-citrulline immunoreactivity, as well as in situ hybridization with a probe specific for Acheta NO synthase were used to localize NO-producing cells. Combining these three approaches we clearly establish that mushroom body interneurons synthesize NO. Furthermore, we demonstrate that experimental interventions known to upregulate neuroblast proliferation modulate NO production: rearing crickets in an enriched sensory environment induces an upregulation of Acheta NO synthase mRNA, and unilateral electrical stimulation of the antennal nerve results in increased L-citrulline immunoreactivity in the corresponding mushroom body. The present study demonstrates that neural activity modulates progenitor cell proliferation and regulates NO production in brain structures where neurogenesis occurs in the adult insect. Our results also demonstrate the stimulatory effect of NO on mushroom body neuroblast proliferation. Altogether, these data strongly suggest a key role for NO in environmentally induced neurogenesis.

The vestibular semicircular canals are internal sensors that signal the magnitude, direction, and temporal properties of angular head motion. Fluid mechanics within the 3-canal labyrinth code the direction of movement and integrate angular acceleration stimuli over time. Directional coding is accomplished by decomposition of complex angular accelerations into 3 biomechanical components-one component exciting each of the 3 ampullary organs and associated afferent nerve bundles separately. For low-frequency angular motion stimuli, fluid displacement within each canal is proportional to angular acceleration. At higher frequencies, above the lower corner frequency, real-time integration is accomplished by viscous forces arising from the movement of fluid within the slender lumen of each canal. This results in angular velocity sensitive fluid displacements. Reflecting this, a subset of afferent fibers indeed report angular acceleration to the brain for low frequencies of head movement and report angular velocity for higher frequencies. However, a substantial number of afferent fibers also report angular acceleration, or a signal between acceleration and velocity, even at frequencies where the endolymph displacement is known to follow angular head velocity. These non-velocity-sensitive afferent signals cannot be attributed to canal biomechanics alone. The responses of non-velocity-sensitive cells include a mathematical differentiation (first-order or fractional) imparted by hair-cell and/or afferent complexes. This mathematical differentiation from velocity to acceleration cannot be attributed to hair cell ionic currents, but occurs as a result of the dynamics of synaptic transmission between hair cells and their primary afferent fibers. The evidence for this conclusion is reviewed below.

The synapsins are presynaptic membrane-associated proteins involved in neurotransmitter release. They are differentially expressed in tissues and cells of the central and peripheral nervous system. In vestibular end organs of mammals, synapsin I-like immunoreactivity has been reported in efferent and afferent terminals and in afferent nerve calyces surrounding type I hair cells. In addition, synapsin I has recently been described in several non-neural cell lines. The present study was conducted to locate synapsin-like immunoreactivity in the neuronal and non-neuronal cells of the fish crista ampullaris, to examine the possibility that the non-neuronal sensory receptor cells express synapsins in vivo. Synapsin-like immunostaining was visualized by immunofluorescence detection in wholemounts of the toadfish crista ampullaris using multiphoton laser scanning microscopy and by electron microscopic visualization of post-embedding immunogold labeling. The results demonstrate that synapsin-like immunoreactivity is present in vestibular hair cells and efferent boutons of the toadfish crista ampullaris. Afferent endings are not labeled. Staining in hair cells is not associated with the synaptic ribbons, suggesting that there is an additional, non-synaptic role for the synapsins in some non-neuronal cells of vertebrates. Moreover, while the cristae of amniote and anamniote species share many functional attributes, differences in their synaptic vesicle-associated protein profiles appear to reflect their disparate hair cell populations.

The time course and extent of adaptation in semicircular canal hair cells was compared to adaptation in primary afferent neurons for physiological stimuli in vivo to study the origins of the neural code transmitted to the brain. The oyster toadfish, Opsanus tau, was used as the experimental model. Afferent firing-rate adaptation followed a double-exponential time course in response to step cupula displacements. The dominant adaptation time constant varied considerably among afferent fibers and spanned six orders of magnitude for the population ( approximately 1 ms to >1,000 s). For sinusoidal stimuli (0.1-20 Hz), the rapidly adapting afferents exhibited a 90 degrees phase lead and frequency-dependent gain, whereas slowly adapting afferents exhibited a flat gain and no phase lead. Hair-cell voltage and current modulations were similar to the slowly adapting afferents and exhibited a relatively flat gain with very little phase lead over the physiological bandwidth and dynamic range tested. Semicircular canal microphonics also showed responses consistent with the slowly adapting subset of afferents and with hair cells. The relatively broad diversity of afferent adaptation time constants and frequency-dependent discharge modulations relative to hair-cell voltage implicate a subsequent site of adaptation that plays a major role in further shaping the temporal characteristics of semicircular canal afferent neural signals.

The vestibular semicircular canals respond to angular acceleration that is integrated to angular velocity by the biofluid mechanics of the canals and is the primary origin of afferent responses encoding velocity. Surprisingly, some afferents actually report angular acceleration. Our data indicate that hair-cell/afferent synapses introduce a mathematical derivative in these afferents that partially cancels the biomechanical integration and results in discharge rates encoding angular acceleration. We examined the role of convergent synaptic inputs from hair cells to this mathematical differentiation. A significant reduction in the order of the differentiation was observed for low-frequency stimuli after gamma-aminobutyric acid type B receptor antagonist administration. Results demonstrate that gamma-aminobutyric acid participates in shaping the temporal dynamics of afferent responses.

We identified the previously unknown structures of ribosylated imidazoleacetic acids in rat, bovine, and human tissues to be imidazole-4-acetic acid-ribotide (IAA-RP) and its metabolite, imidazole-4-acetic acid-riboside. We also found that IAA-RP has physicochemical properties similar to those of an unidentified substance(s) extracted from mammalian tissues that interacts with imidazol(in)e receptors (I-Rs). ["Imidazoline," by consensus (International Union of Pharmacology), includes imidazole, imidazoline, and related compounds. We demonstrate that the imidazole IAA-RP acts at I-Rs, and because few (if any) imidazolines exist in vivo, we have adopted the term "imidazol(in)e-Rs."] The latter regulate multiple functions in the CNS and periphery. We now show that IAA-RP (i) is present in brain and tissue extracts that exhibit I-R activity; (ii) is present in neurons of brainstem areas, including the rostroventrolateral medulla, a region where drugs active at I-Rs are known to modulate blood pressure; (iii) is present within synaptosome-enriched fractions of brain where its release is Ca(2+)-dependent, consistent with transmitter function; (iv) produces I-R-linked effects in vitro (e.g., arachidonic acid and insulin release) that are blocked by relevant antagonists; and (v) produces hypertension when microinjected into the rostroventrolateral medulla. Our data also suggest that IAA-RP may interact with a novel imidazol(in)e-like receptor at this site. We propose that IAA-RP is a neuroregulator acting via I-Rs.

The present study was conducted to visualize the ultrastructural features of vestibular efferent boutons in the oyster toadfish, Opsanus tau. The crista ampullaris of the horizontal semicircular canal was processed for and examined by routine transmission electron microscopy. The results demonstrate that such boutons vary in size and shape, and contain a heterogeneous population of lucent vesicles with scattered dense core vesicles. Efferent contacts with hair cells are characterized by local vesicle accumulations in the presynaptic terminal and a subsynaptic cistern in the postsynaptic region of the hair cell. Serial efferent to hair cell to afferent synaptic arrangements are common, particularly in the central portion of the crista. However, direct contacts between efferent terminals and afferent neurites were not observed in our specimens. The existence of serial synaptic contacts, often with a row of vesicles in the efferent boutons lining the efferent-afferent membrane apposition, suggests that the efferent influence on the crista may involve both synaptic and nonsynaptic, secretory mechanisms. Further, it is suggested that differences in more subtle aspects of synaptic architecture and/or transmitter and receptor localization and interaction may render the efferent innervation of the peripheral crista less effective in influencing sensory processing.

Although gamma-aminobutyric acid (GABA) and glutamate are known to be present in the vestibular sensory epithelia of a variety of species, the functional relationship between these two transmitters is not clear. The present study addresses the three-dimensional spatial distribution of GABA and glutamate immunoreactivity in the vestibular labyrinth of the oyster toadfish by using whole end organs labeled by immunofluorescence with monoclonal anti-GABA and/or antiglutamate antibodies and visualized as whole mounts by multiphoton confocal microscopy. We find glutamate-immunoreactive hair cells present throughout the sensory epithelium. In contrast, prominent GABA immunoreactivity is restricted to a small population of hair cells located in the central region of the crista. Double immunofluorescence reveals two distinct staining patterns in GABA-labeled hair cells. Most ( approximately 80%) GABA-labeled cells show trace levels of glutamate, appropriate for the metabolic/synthetic role of cytoplasmic glutamate. The remainder of the GABA-stained cells contain substantial levels of both GABA and glutamate, suggesting transmitter colocalization. In the toadfish utricle, glutamatergic hair cells are present throughout the macula. GABA-immunoreactive hair cells follow the arc of the striola, and most GABA-labeled receptor cells coexpress glutamate. The localization of GABA was explored in other species as well. In the pigeon, GABAergic hair cells are present throughout the crista ampullaris. Our findings demonstrate that multiple, neurochemically distinct types of hair cells are present in vestibular sensory epithelia. These observations, together with the excitatory activity generally associated with 8th nerve afferent fibers, strongly suggest that GABA serves an important, specific, and complex role in determining primary afferent response dynamics., (Copyright 2004 Wiley-Liss, Inc.)

The cellular and subcellular localization of L-citrulline was analyzed in the adult rat brain and compared with that of traditional markers for the presence of nitric oxide synthase. Light, transmission electron, and confocal laser scanning microscopy were used to study tissue sections processed for immunocytochemistry employing a monoclonal antibody against L-citrulline or polyclonal anti-neuronal nitric oxide synthase sera, and double immunofluorescence to detect neuronal nitric oxide synthase and L-citrulline co-localization. The results demonstrate that the same CNS regions and cell types are labeled by neuronal nitric oxide synthase polyclonal antisera and L-citrulline monoclonal antibodies, using both immunocytochemistry and immunofluorescence. Short-term pretreatment with a nitric oxide synthase inhibitor reduces L-citrulline immunostaining, but does not affect neuronal nitric oxide synthase immunoreactivity. In the vestibular brainstem, double immunofluorescence studies show that many, but not all, neuronal nitric oxide synthase-positive cells co-express L-citrulline, and that local intracellular patches of intense L-citrulline accumulation are present in some neurons. Conversely, all L-citrulline-labeled neurons co-express neuronal nitric oxide synthase. Cells expressing neuronal nitric oxide synthase alone are interpreted as neurons with the potential to produce nitric oxide under other stimulus conditions, and the subcellular foci of enhanced L-citrulline staining are viewed as intracellular sites of nitric oxide production. This interpretation is supported by ultrastructural observations of subcellular foci with enhanced L-citrulline and/or neuronal nitric oxide synthase staining that are located primarily at postsynaptic densities and portions of the endoplasmic reticulum. We conclude that nitric oxide is produced and released at focal sites within neurons that are identifiable using L-citrulline as a marker., (Copyright 2002 IBRO)