Ch Lai

Secretin as a neurohypophysial factor regulating body water homeostasis

Hypothalamic magnocellular neurons express either one of the neurohypophysial hormones, vasopressin or oxytocin, along with different neuropeptides or neuromodulators. Axonal terminals of these neurons are generally accepted to release solely the two hormones but not others into the circulation. Here, we show that secretin, originally isolated from upper intestinal mucosal extract, is present throughout the hypothalamo–neurohypophysial axis and that it is released from the posterior pituitary under plasma hyperosmolality conditions. In the hypothalamus, it stimulates vasopressin expression and release. Considering these findings together with our previous findings that show a direct effect of secretin on renal water reabsorption, we propose here that secretin works at multiple levels in the hypothalamus, pituitary, and kidney to regulate water homeostasis. Findings presented here challenge previous understanding regarding the neurohypophysis and could provide new concepts in treating disorders related to osmoregulation.

Fos expression in otolith-related brainstem neurons of postnatal rats following off-vertical axis rotation

To determine the critical time of responsiveness of developing otolith organ‐related brainstem neurons and their distribution, Fos protein expression in response to off‐vertical axis rotations (OVAR) was mapped in conscious Sprague Dawley rats from P5 to adulthood. OVAR was used to activate sequentially all utricular hair cells per 360° revolution. We detected the coding of horizontal head positions in otolith organ‐related neurons within the vestibular nucleus as early as P7. In the vestibular nuclear complex and its subgroups, the density of Fos‐immunoreactive (Fos‐ir) neurons increased steadily with age and reached the adult level by P21. In both labyrinthectomized rats subjected to OVAR and normal rats kept stationary, labeled neurons were found sporadically in the aforementioned brain regions in each age group, confirming that Fos labeling observed in neurons of normal experimental rats subjected to OVAR was due to otolith organ stimulation. Whereas OVAR‐induced Fos‐ir neurons were also first observed in vestibular‐related brain areas, such as the prepositus hypoglossal nucleus, gigantocellular reticular nucleus, and locus coeruleus, of normal experimental rats at P7, those in the inferior olive were observed only from P14 onward. This indicates the unique maturation time of inferior olivary neurons in gravity‐related spatial coding. In general, age‐dependent increase in OVAR‐induced Fos‐ir neurons was observed in brain areas that received otolith inputs. The locus coeruleus was exceptional in that prominent OVAR‐induced Fos‐ir neuronal number did not change with maturation, and this was well above the low but significant number of Fos‐ir neurons in control preparations. Taken together, our results suggest that neuronal subpopulations within the developing network of the horizontal otolith system provide an anatomical basis for the postnatal development of otolith organ‐related sensorimotor functions. J. Comp. Neurol. 470:282–296, 2004.

Quantitative study of the coexpression of Fos and N-methyl-D aspartate (NMDA) receptor subunits in otolith-related vestibular nuclear neurons of rats

The expression of NMDA receptor subunits (NR1 and NR2A/B) was demonstrated immunocytochemically in otolith‐related neurons within the vestibular nuclear complex and its subnuclei of conscious Sprague‐Dawley adult rats. All experimental animals were subjected to constant velocity off‐vertical axis rotation (OVAR). The rotating gravity vector during OVAR sequentially activates hair cells on all sectors of the utricular maculae; neurons so activated within the vestibular nuclei were denoted by the expression of Fos protein. Control animals, i.e., labyrinthectomized rats subjected to OVAR and normal rats that remained stationary, showed only a few sporadically scattered labeled neurons. In the brainstem of normal rats subjected to OVAR, a high density of Fos‐immunoreactive (Fos‐ir) neurons was found in the vestibular nuclear complex (namely, spinal vestibular nucleus, SpVe; medial vestibular nucleus, Mve; superior vestibular nucleus, SuVe) and subnuclei (namely, group x and group y), whereas a lower density was found in the lateral vestibular nucleus (LVe). A double‐immunofluorescence study indicated that both NR1 and NR2A/B subunits were highly expressed in Fos‐ir neurons within the vestibular nuclei. Fos/NR1 or Fos/NR2A/B double‐labeled neurons constitute over three‐quarters of the total number of Fos‐ir neurons in SpVe, MVe, LVe, SuVe, and groups x and y. Our findings suggest that NMDA‐type ionotropic glutamate receptors play a key role in the OVAR‐induced neuronal activation of the vestibular nuclei, thus providing a morphological basis for further study of glutamatergic central otolith neurons and their involvement in sensorimotor regulation and autonomic functions of rats. J. Comp. Neurol. 460:292–301, 2003.

Spontaneous discharge and response characteristics of central otolith neurons of rats during postnatal development

To study the developmental profile of otolith-related vestibular nuclear neurons, their spontaneous activities and response dynamics were examined in decerebrate rats aged seven, 14, 21 and 84 (adult) days. Extracellular recordings were performed in the lateral and descending vestibular nucleus of animals held at the stationary position in the earth-horizontal or subjected to constant velocity off-vertical axis rotation, which selectively stimulates the otolith receptors. All neurons displayed sinusoidal position-dependent modulation in discharge rate, indicating their capability in coding spatial information during low-frequency head movement. Some neurons showed a full-cycle response to off-vertical axis rotation (non-clipped), while other neurons were silenced in discharge during parts of each rotary cycle (clipped). In seven-day-old rats, three-quarters of the responsive neurons sampled were clipped and the proportion progressively decreased to less than one-quarter in adult rats. In each age group, the clipped neurons discharged in approximately 60% of the stimulus cycle. Response gains of the neurons increased with age, reaching a plateau from 21 days of age for clipped neurons and 14 days for non-clipped neurons. The clipped neurons demonstrated higher response gains than the non-clipped neurons at or beyond 21 days of age. Spontaneous activities of the neurons at the stationary and earth-horizontal positions were analysed in relation to their response gains; a positive correlation was observed from 14 days of age onwards. Both types of neurons showed progressive increase in spontaneous activity as the rats matured, though the clipped neurons exhibited significantly lower resting rates than the non-clipped neurons at each of the age groups studied. Some neurons that responded to off-vertical axis rotation were not spontaneously active at the stationary position, but the proportion of these decreased significantly with age. The coefficient of variation of each age group showed a bimodal distribution, thereby allowing spontaneously active neurons to be assigned as regular or irregular. Though the vast majority of both the clipped and non-clipped neurons showed irregular discharge patterns at seven days of age, the overall population became more regular as the rats matured. Irregular neurons of young rats exhibited phase-stable and phase-shift responses, while those of older rats showed only the phase-stable response. This distinction was not observed amongst regular neurons over the ages studied. Our results reveal features of central otolith neurons that can be taken as signs of maturation during the first three postnatal weeks. These neuronal features provide the framework for the analysis of behaviours mediated by the otolith system during postnatal maturation.

Developmental expression of NMDA and AMPA receptor subunits in vestibular nuclear neurons that encode gravity-related horizontal orientations.

We examined the expression profile of subunits of ionotropic glutamate receptors [N‐methyl‐D‐aspartate (NMDA) and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐proprionate (AMPA)] during postnatal development of connectivity in the rat vestibular nucleus. Vestibular nuclear neurons were functionally activated by constant velocity off‐vertical axis rotation, a strategy to stimulate otolith organs in the inner ear. These neurons indicated Fos expression as a result. By immunodetection for Fos, otolith‐related neurons that expressed NMDA/AMPA receptor subunits were identified as early as P7, and these neurons were found to increase progressively up to adulthood. Although there was developmental invariance in the percentage of Fos‐immunoreactive neurons expressing the NR1, NR2A, GluR1, or GluR2/3 subunits, those expressing the NR2B subunit decreased from P14 onward, and those expressing the GluR4 subunit decreased in adults. These double‐immunohistochemical data were corroborated by combined immuno‐/hybridization histochemical data obtained from Fos‐immunoreactive neurons expressing NR2B mRNA or GluR4 mRNA. The staining of both NR2B and GluR4 in the cytoplasm of these neurons decreased upon maturation. The percentage of Fos‐immunoreactive neurons expressing the other ionotropic glutamate receptor subunits (viz. NR1, NR2A, GluR1, and GluR2/3) remained relatively constant throughout postnatal maturation. Triple immunofluorescence further demonstrated coexpression of NR1 and NR2 subunits in Fos‐immunoreactive neurons. Coexpression of NR1 subunit with each of the GluR subunits was also observed among the Fos‐immunoreactive neurons. Taken together, the different expression profiles of ionotropic glutamate receptor subunits constitute the histological basis for glutamatergic neurotransmission in the maturation of central vestibular connectivity for the coding of gravity‐related horizontal head movements. J. Comp. Neurol. 508:343–364, 2008.

Neuronal response sensitivity to bidirectional off-vertical axis rotations: A dimension of imbalance in the bilateral vestibular nuclei of cats after unilateral labyrinthectomy

In decerebrate cats after acute hemilabyrinthectomy, the response sensitivity of extracellularly recorded vestibular nuclear neurons on the lesioned and labyrinth-intact sides were examined quantitatively during constant velocity off-vertical axis rotations with an aim to elucidate the functional contribution of otolithic inputs to the ipsilateral and contralateral vestibular nuclei. The bidirectional response sensitivity, delta, was determined as the ratio of the gain during clockwise to that during counterclockwise rotations. A continuum of response sensitivity was identified: one-dimensional neurons showed symmetrically bidirectional response patterns, while two-dimensional neurons showed asymmetrically bidirectional patterns that in some cases approached unidirectional patterns with change in velocity. The proportion of two-dimensional neurons was significantly increased after acute hemilabyrinthectomy. Two-dimensional neurons that responded only to one direction of rotation in at least one of the velocities tested were described as unidirectional neurons. This unidirectional response pattern was observed in one-third of the entire neuronal population studied, but not in cats with both labyrinths intact, thus suggesting that such prominent broadly tuned responses are normally masked by converging otolithic inputs from the contralateral side. These neurons were found in higher proportion on the lesioned side than on the labyrinth-intact side. Among the 70% of unidirectional neurons that exhibited bidirectional response at some velocities and unidirectional response at others, prominent shifts in delta values (i.e. between 0/infinity and finite values) with velocity can be computed for each neuron. The shifts in delta values correlated with large shifts in the response dynamics and spatial orientation as the response pattern changed with velocity. The response orientations of the unidirectional neurons pointed in all directions on the horizontal plane. When all the two-dimensional neurons (i.e. both the unidirectionally and bidirectionally responsive ones) were pooled, imbalances in the distribution of the response orientations and in response gain were found between the ipsilateral-side-down/head-down half-circle and the contralateral-side-down/head-up half-circle on the labyrinth-intact side, but not on the lesioned side. These results, derived from spatiotemporal processing of gravitational signals, reveal a novel dimension of imbalance between neuronal populations in the two vestibular nuclear complexes after acute lesion of one labyrinth. This feature would provide, on the one hand, deranged cues of spatial orientation and direction during slow head excursions and, on the other, a framework for the dynamic behavioral deficits associated with hemilabyrinthectomy.

Corticofugal modulation of acoustically induced Fos expression in the rat auditory pathway

To investigate the corticofugal modulation of acoustic information ascending through the auditory pathway of the rat, immunohistochemical techniques were used to study the functional expression of Fos protein in neurons. With auditory stimulation at different frequencies, Fos expression in the medial geniculate body (MGB), inferior colliculus (IC), superior olivary complex, and cochlear nucleus was examined, and the extent of Fos expression on the two sides was compared. Strikingly, we found densely Fos‐labeled neurons in all divisions of the MGB after both presentation of an auditory stimulus and administration of a γ‐aminobutyric acid type A (GABAA) antagonist (bicuculline methobromide; BIM) to the auditory cortex. The location of Fos‐labeled neurons in the ventral division (MGv) after acoustic stimulation at different frequencies was in agreement with the known tonotopic organization. That no Fos‐labeled neurons were found in the MGv with acoustic stimuli alone suggests that the transmission of ascending thalamocortical information is critically governed by corticofugal modulation. The dorsal (DCIC) and external cortices (ECIC) of the IC ipsilateral to the BIM‐injected cortex showed a significantly higher number of Fos‐labeled neurons than the contralateral IC. However, no difference in the number of Fos‐labeled neurons was found between the central nucleus of the IC on either side, indicating that direct corticofugal modulation occurs only in the ECIC and DCIC. Further investigations are needed to assess the functional implications of the morphological differences observed between the descending corticofugal projections to the thalamus and the IC. J. Comp. Neurol. 501:509–525, 2007.

Expression of Trk receptors in otolith-related neurons in the vestibular nucleus of rats.

The expression of the three Trk receptors (TrkA, TrkB, and TrkC) in otolith-related neurons within the vestibular nuclei of adult Sprague-Dawley rats was examined immunohistochemically. Conscious animals were subjected to sinusoidal linear acceleration along either the anterior-posterior (AP) or interaural (IA) axis on the horizontal plane. Neuronal activation was defined by Fos expression in cell nuclei. Control animals, viz labyrinthectomized rats subjected to stimulation and normal rats that remained stationary, showed only a few sporadically scattered Fos-labeled neurons. Among experimental rats, the number of Fos-labeled neurons and their distribution pattern in each vestibular subnucleus in animals stimulated along the antero-posterior axis were similar to those along the interaural axis. No apparent topography was observed among neurons activated along these two directions. Only about one-third of the Trk-immunoreactive neurons in the vestibular nucleus expressed Fos. Double-labeled Fos/TrkA, Fos/TrkB and Fos/TrkC neurons constituted 85-98% of the total number of Fos-labeled neurons in vestibular nuclear complex and its subgroups x and y. Our findings suggest that Trk receptors and their cognate neurotrophins in central otolith neurons may contribute to the modulation of gravity-related spatial information during horizontal head movements.

Maturation of otolith-related brainstem neurons in the detection of vertical linear acceleration in rats.

To investigate the critical maturation time of otolith‐related neurons in processing vertical orientations, rats (postnatal day 4 to adults) were studied for functional activation of c‐fos expression in brainstem neurons by immuno‐/hybridization histochemistry. Conscious rats were subjected to sinusoidal linear acceleration along the vertical plane. Labyrinthectomized and/or stationary controls showed only sporadically scattered Fos‐labeled neurons in the vestibular nuclei, confirming an otolithic origin of c‐fos expression. Functionally activated Fos expression in neurons of the medial and spinal vestibular nuclei and group x were identifiable by P7 and those in group y by P9. A small number of Fos‐labeled neurons characterized by small soma size were found in the ventral part of lateral vestibular nucleus by P9. Other vestibular‐related areas such as prepostitus hypoglossal nucleus, gigantocellular reticular nucleus and locus coeruleus of normal experimental rats showed functionally activated c‐fos expression at P7. Neurons in dorsal medial cell column and beta subnucleus of the inferior olive only showed functionally activated c‐fos expression by the second postnatal week. These findings revealed a unique critical maturation time for each of the vestibular‐related brainstem areas in the recognition of gravity‐related vertical head orientations. By mapping the three‐dimensional distribution of Fos‐immunoreactive neurons, we found an even distribution of otolith‐related neurons within the spinal vestibular nucleus in groups x and y but a clustered distribution in the middle–lateral–ventral part of the medial vestibular nucleus. Taken together, our findings reveal the developmental profile of neuronal subpopulations within the vertical otolith system, thereby providing an anatomical basis for postnatal coding of gravity‐related vertical head movements.

Properties of otolith-related vestibular nuclear neurons in response to bidirectional off-vertical axis rotation of the rat

In decerebrate rats, the responses of tilt-sensitive neurons in the lateral and descending vestibular nuclei were studied during constant velocity 10 degrees off-vertical axis rotations (OVAR) in the clockwise (VW) and counterclockwise (CCW) directions. Seventy three otolith-related units showed a sinusoidal position-dependent discharge modulation to OVAR of both directions; 20 of these showed clipped firing rates in parts of a 360 degree OVAR cycle. With increase in the velocity of rotation (1.75-15 degrees/s), one group of units (n = 36) showed a stable ratio of bidirectional response sensitivity and symmetric response magnitudes to CW and CCW rotations. These units showed gain tuning ratios similar to those of narrowly spatiotemporal-tuned neurons. The other group of OVAR responsive units (n = 13) exhibited velocity-variable and asymmetric bidirectional response sensitivities. Their gain tuning ratios were similar to those of broadly spatiotemporal-tuned neurons. For units with velocity-stable and symmetric bidirectional response sensitivity as well as gain tuning ratio of the narrowly spatiotemporal-tuned neurons, their response gains remained stable with velocity. Some showed stable response phase lead or lag with velocity increase while others showed progressive shifts from response lead of 13 degrees to response lag of -25 degrees. The best response orientations of these units with velocity-stable and symmetric bidirectional response sensitivity were found to point in all directions on the place of rotation. The functional significance of these tilt- and OVAR-sensitive central otolith neurons is discussed.