Expression of ZO1, vimentin, pan-cadherin and AGTR1 in tanycyte-like cells of the sulcus medianus organum.

Tanycytes are a specialized ependymal lining of brain ventricles with exceptional features of having long basal processes and junctional complexes between cell bodies. These tanycytes are present at the regions of circumventricular organs (CVOs) which possess common morphological and functional features enabling them to be described as the brain windows where the barrier systems have special properties. Previous studies detailed seven of these CVOs but little information is available regarding another putative site at the rostral part of the median sulcus of the 4th ventricle, or the sulcus medianus organum (SMO). Here we performed a pilot immunohistochemical study to support earlier observations suggesting the SMO as a novel CVO. We labeled rat brain with ZO1, vimentin, pan-cadherin and angiotensin II type 1 receptors markers which showed a morphologically distinct population of cells at the region of the SMO similar to tanycytes present in the median eminence, a known CVO. These cells had basal processes reaching the deeply seated blood vessels while the caudal part of the median sulcus did not show similar long cellular extensions. We concluded that tanycyte-like cells are present in the SMO in a pattern resembling that of other CVOs where the strategic location of the SMO is probably for signal integration between brainstem nuclei and the rostrally located neuronal centers.

Identification of proliferative progenitors associated with prominent postnatal growth of the pons.

The pons controls crucial sensorimotor and autonomic functions. In humans, it grows sixfold postnatally and is a site of paediatric gliomas; however, the mechanisms of pontine growth remain poorly understood. We show that the murine pons quadruples in volume postnatally; growth is fastest during postnatal days 0-4 (P0-P4), preceding most myelination. We identify three postnatal proliferative compartments: ventricular, midline and parenchymal. We find no evidence of postnatal neurogenesis in the pons, but each progenitor compartment produces new astroglia and oligodendroglia; the latter expand 10- to 18-fold postnatally, and are derived mostly from the parenchyma. Nearly all parenchymal progenitors at P4 are Sox2(+)Olig2(+), but by P8 a Sox2(-) subpopulation emerges, suggesting a lineage progression from Sox2(+) 'early' to Sox2(-) 'late' oligodendrocyte progenitor. Fate mapping reveals that >90% of adult oligodendrocytes derive from P2-P3 Sox2(+) progenitors. These results demonstrate the importance of postnatal Sox2(+)Olig2(+) progenitors in pontine growth and oligodendrogenesis.

Commissural nucleus of the solitary tract regulates the antihypertensive effects elicited by moxonidine.

The rostral ventrolateral medulla (RVLM) contains the presympathetic neurons involved in cardiovascular regulation that has been implicated as one of the most important central sites for the antihypertensive action of moxonidine (an α2-adrenergic and imidazoline agonist). Here, we sought to evaluate the cardiovascular effects produced by moxonidine injected into another important brainstem site, the commissural nucleus of the solitary tract (commNTS). Mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic nerve activity (sSNA) and activity of putative sympathoexcitatory vasomotor neurons of the RVLM were recorded in conscious or urethane-anesthetized, and artificial ventilated male Wistar rats. In conscious or anesthetized rats, moxonidine (2.5 and 5 nmol/50 nl) injected into the commNTS reduced MAP, HR and sSNA. The injection of moxonidine into the commNTS also elicited a reduction of 28% in the activity of sympathoexcitatory vasomotor neurons of the RVLM. To further assess the notion that moxonidine could act in another brainstem area to elicit the antihypertensive effects, a group with electrolytic lesions of the commNTS or sham and with stainless steel guide-cannulas implanted into the 4th V were used. In the sham group, moxonidine (20 nmol/1 µl) injected into 4th V decreased MAP and HR. The hypotension but not the bradycardia produced by moxonidine into the 4th V was reduced in acute (1 day) commNTS-lesioned rats. These data suggest that moxonidine can certainly act in other brainstem regions, such as commNTS to produce its beneficial therapeutic effects, such as hypotension and reduction in sympathetic nerve activity.

Expression of doublecortin, a neuronal migration protein, in unipolar brush cells of the vestibulocerebellum and dorsal cochlear nucleus of the adult rat.

Doublecortin (DCX) is a microtubule-associated protein that is critical for neuronal migration and the development of the cerebral cortex. In the adult, it is expressed in newborn neurons in the subventricular and subgranular zones, but not in the mature neurons of the cerebral cortex. By contrast, neurogenesis and neuronal migration of cells in the cerebellum continue into early postnatal life; migration of one class of cerebellar interneuron, unipolar brush cells (UBCs), may continue into adulthood. To explore the possibility of continued neuronal migration in the adult cerebellum, closely spaced sections through the brainstem and cerebellum of adult (3-16 months old) Sprague-Dawley rats were immunolabeled for DCX. Neurons immunoreactive (ir) to DCX were present in the granular cell layer of the vestibulocerebellum, most densely in the transition zone (tz), the region between the flocculus (FL) and ventral paraflocculus (PFL), as well as in the dorsal cochlear nucleus (DCN). These DCX-ir cells had the morphological appearance of UBCs with oval somata and a single dendrite ending in a brush. There were many examples of colocalization of DCX with Eps8 or calretinin, UBC markers. We also identified DCX-ir elements along the fourth ventricle and its lateral recess that had labeled somata but lacked the dendritic structure characteristic of UBCs. Labeled UBCs were seen in nearby white matter. These results suggest that there may be continued neurogenesis and/or migration of UBCs in the adult. Another possibility is that UBCs maintain DCX expression even after migration and maturation, reflecting a role of DCX in adult neuronal plasticity in addition to a developmental role in migration.

Regulatory volume increase in epithelial cells isolated from the mouse fourth ventricle choroid plexus involves Na(+)-H(+) exchange but not Na(+)-K(+)-2Cl(-) cotransport.

The aim of this study was to determine the ability of choroid plexus epithelial cells to volume regulate when exposed to hypertonic solutions, and furthermore to identify the ion transporters involved in any volume regulation. Experiments were performed on cells freshly isolated, using the enzyme dispase, from the mouse fourth ventricle choroid plexus. Cell volume was measured using a video-imaging method. Cells used in this study were all of a similar morphology and had a mean volume of 0.71pl. Cells shrank when superfused with hypertonic solutions to a minimum relative cell volume of 0.84+/-0.01 (n=8) in 3min. They then exhibited a regulatory volume increase (RVI) to reach a relative volume of 0.92+/-0.02 over the following 12min. The RVI was HCO(3)(-)-dependent, that is it was not observed in hepes-buffered solutions. A post-regulatory volume decrease RVI (post-RVD RVI) was also observed in cells following exposure to hypotonic solutions. The RVI and post-RVD RVI were inhibited by 10microM 5-(N-ethyl-N-isopropyl) amiloride or 10microM 5-(N-methyl-N-isobutyl) amiloride, both selective inhibitors of Na(+)-H(+) exchange (NHE). They were also inhibited by the anion transport inhibitor 100microM 2,2'-(1,2-ethenediyl) bis (5-isothiocyanatobenzenesulfonic acid). The Na(+)-K(+)-2Cl(-) cotransporter inhibitor, 10microM bumetanide, was without effect on either the RVI or the post-RVD RVI. The data indicate that NHE, probably in combination with Cl(-)-HCO(3)(-) exchangers, contributes to RVI in choroid plexus epithelial cells.

Circumventricular organs: a novel site of neural stem cells in the adult brain.

Neurogenesis in the adult mammalian nervous system is now well established in the subventricular zone of the anterolateral ventricle and subgranular zone of the hippocampus. In these regions, neurons are thought to arise from neural stem cells, identified by their expression of specific intermediate filament proteins (nestin, vimentin, GFAP) and transcription factors (Sox2). In the present study, we show that in adult rat and mouse, the circumventricular organs (CVOs) are rich in nestin+, GFAP+, vimentin+ cells which express Sox2 and the cell cycle-regulating protein Ki67. In culture, these cells proliferate as neurospheres and express neuronal (doublecortin+, beta-tubulin III+) and glial (S100beta+, GFAP+, RIP+) phenotypic traits. Further, our in vivo studies using bromodeoxyuridine show that CVO cells proliferate and undergo constitutive neurogenesis and gliogenesis. These findings suggest that CVOs may constitute a heretofore unknown source of stem/progenitor cells, capable of giving rise to new neurons and/or glia in the adult brain.

Melanin-concentrating hormone (MCH) immunoreactivity in non-neuronal cells within the raphe nuclei and subventricular region of the brainstem of the cat.

Neurons that utilize melanin-concentrating hormone (MCH) as a neuromodulator are localized within the postero-lateral hypothalamus and zona incerta. These neurons project diffusely throughout the central nervous system and have been implicated in critical physiological processes such as energy homeostasis and sleep. In the present report, we examined the distribution of MCH immunoreactivity in the brainstem of the cat. In addition to MCH+ axons, we found MCH-immunoreactive cells that have not been previously described either in the midbrain raphe nuclei or in the periaqueductal and periventricular areas. These MCH+ cells constituted: 1. ependymal cells that lined the fourth ventricle and aqueduct, 2. ependymal cells with long basal processes that projected deeply into the subventricular (subaqueductal) parenchyma, and, 3. cells in subventricular regions and the midbrain raphe nuclei. The MCH+ cells in the midbrain raphe nuclei were closely related to neuronal processes of serotonergic neurons. Utilizing Neu-N and GFAP immunohistochemistry we determined that the preceding MCH+ cells were neither neurons nor astrocytes. However, we found that vimentin, an intermediate-filament protein that is used as a marker for tanycytes, was specifically co-localized with MCH in these cells. We conclude that MCH is present in tanycytes whose processes innervate the midbrain raphe nuclei and adjacent subependymal regions. Because tanycytes are specialized cells that transport substances from the cerebrospinal fluid (CSF) to neural parenchyma, we suggest that MCH is absorbed from the CSF by tanycytes and subsequently liberate to act upon neurons of brainstem nuclei.

A TAT-modified fusion protein efficiently penetrates mouse hypoglossal nuclei from transduced ependyma.

Future gene therapy for brainstem variant amyotrophic lateral sclerosis may be technically difficult if gene therapy vectors are injected near vital cardiorespiratory centers or if large portions of the tongue and pharyngeal muscles must be peripherally injected for retrograde transport of vectors to motor neurons. In this study we show that it is possible to deliver recombinant proteins to the hypoglossal nuclei without brainstem or muscle injections, by taking advantage of enhanced uptake of fusion proteins containing the protein transduction domain from the human immunodeficiency virus TAT protein. Adenoviral vectors expressing either TAT-modified or native beta-glucuronidase (beta-gluc) were injected into the lateral cerebral ventricles of mice, transducing ventricular epithelium down to the level of the obex in the brainstem. There was significant uptake into the hypoglossal nuclei of TAT-modified but not native beta-glucuronidase. The TAT-modified beta-gluc appeared to encompass half or more of the hypoglossal nuclei as visualized by retrograde labeling with cholera toxin subunit b in adjacent sections. TAT-modification of gene products may allow a relatively non-invasive approach to brainstem gene therapy via cerebroventricular injection.

Fibroblast growth factor 2 induces loss of adult oligodendrocytes and myelin in vivo.

Oligodendrocytes are the myelin-forming cells of the CNS and are lost in demyelinating diseases such as multiple sclerosis (MS). A role for fibroblast growth factor 2 (FGF2) has been proposed in the pathogenesis of demyelination and the failure of remyelination in experimental models of MS. However, the in vivo effects of FGF2 on oligodendrocytes and oligodendrocyte progenitors (OPCs) in the adult CNS had not previously been determined. To address this, FGF2 was delivered into the cerebrospinal fluid (CSF) of the IVth ventricle and its actions were examined on the anterior medullary velum (AMV), a thin tissue that partly roofs the IVth ventricle and is bathed by CSF. FGF2 was administered twice daily for 3 days and AMV were analysed using immunohistochemical labelling; saline was administered in controls. The results show that raised FGF2 induces severe disruption of mature oligodendrocytes and a marked loss of myelin. At the same time, FGF2 treatment resulted in the aberrant accumulation of immature oligodendrocytes with a premyelinating phenotype, together with NG2-expressing OPCs. Axons are patent within demyelinated lesions, and they are contacted but not ensheathed by surviving oligodendrocytes, newly formed premyelinating oligodendrocytes and OPCs. These results demonstrate that raised FGF2 induces demyelination in the adult CNS, and support a role for FGF2 in the pathogenesis of demyelination and regulation of remyelination in MS.

Glucose does not activate nonadrenergic, noncholinergic inhibitory neurons in the rat stomach.

We reported previously that intravenously administered d-glucose acts in the central nervous system to inhibit gastric motility induced by hypoglycemia in anesthetized rats. The purpose of this study was to determine whether this effect is due to inhibition of dorsal motor nucleus of the vagus (DMV) cholinergic motoneurons, which synapse with postganglionic cholinergic neurons, or to excitation of DMV cholinergic neurons, which synapse with postganglionic nonadrenergic, noncholinergic (NANC) neurons, particularly nitrergic neurons. Three approaches were employed: 1) assessment of the efficacy of d-glucose-induced inhibition of gastric motility in hypoglycemic rats with and without inhibition of nitric oxide synthase [10 mg/kg iv nitro-l-arginine methyl ester (l-NAME)], 2) assessment of the efficacy of intravenous bethanechol (30 mug.kg(-1).min(-1)) to stimulate gastric motility in hypoglycemic rats during the time of d-glucose-induced inhibition of gastric motility, and 3) determination of c-Fos expression in DMV neurons after intravenous d-glucose was administered to normoglycemic rats. Results obtained demonstrated that l-NAME treatment had no effect on d-glucose-induced inhibition of gastric motility; there was no reduction in the efficacy of intravenous bethanechol to increase gastric motility, and c-Fos expression was not induced by d-glucose in DMV neurons that project to the stomach. These findings indicate that excitation of DMV cholinergic motoneurons that synapse with postganglionic NANC neurons is not a significant contributing component of d-glucose-induced inhibition of gastric motility.

Somatostatin in the brain of the cave salamander, Hydromantes genei (Amphibia, Plethodontidae): immunohistochemical localization and biochemical characterization.

The distribution of somatostatin-like immunoreactivity in the brain of the cave salamander Hydromantes genei (Amphibia, Plethodontidae) was investigated by using two distinct antisera raised against somatostatin-14. Most somatostatin-positive cells were detected in the ependymal cell layer surrounding the ventricles. These cells possessed the typical morphological characteristics of tanycytes or radial glial cells. Double-labeling with an antiserum against somatostatin and a monoclonal antibody against glial fibrillary acidic protein showed that somatostatin-immunoreactive cells lining the ventricles also exhibited GFAP-like immunoreactivity. Injection of the neurotracer biocytin into the lateral ventricle revealed that neurons lining the ventricles did not contain somatostatin-like immunoreactivity. In the telencephalon, somatostatin-like immunoreactivity was confined to radial glial cells. In the diencephalon, in addition to somatostatin-immunoreactive cells in the ependyma, positive cell bodies were also found in the periventricular preoptic nucleus, the infundibular nucleus, the epiphysis, and the subcommissural organ. In the metencephalon, positive cell bodies were found in the auricula cerebelli, whereas in the rhombencephalon numerous somatostatin-immunoreactive cells were seen lining the ventricular cavity. Immunoreactive nerve fibers were observed in the hypothalamus-median eminence complex. In the pituitary, a discrete group of somatostatin-positive cells was found in the pars distalis. High-performance liquid chromatography analysis of brain extracts revealed that the immunoreactive material coeluted with somatostatin-14. The present results show that the somatostatin peptidergic system in the brain of the cave salamander has a more simple organization than those described in the brain of frog and other vertebrates. This feature is probably related to the expression of high pedomorphic characters in plethodontids. The distribution of somatostatin-like immunoreactivity suggests that, in the cave salamander, somatostatin may act as a neurotransmitter and/or neuromodulator, a central regulator of fluid homeostasis, and a hypophysiotropic neurohormone.

Implantation of neural stem cells via cerebrospinal fluid into the injured root.

In avulsion injury of the dorsal root, regenerating axons cannot extend through the entry zone, i.e. the transition zone between peripheral and central nervous systems, due to the discontinuity between Schwann cells and astrocytes. We infused neural stem cells through the 4th ventricle in an attempt to enhance axonal growth in injured dorsal roots. Infused stem cells were attached to, and integrated into, the lesion of the root and became associated with axons in the same manner as Schwann cells or perineurial sheath cells in the peripheral nerve, and as astrocytes in the central nerve area. These findings suggest that neural stem cells integrated by infusion through CSF might have a beneficial effect on nerve regeneration by inducing a continuity of Schwann cells and astrocytes at the transition zone.

Dissemination and proliferation of neural stem cells on the spinal cord by injection into the fourth ventricle of the rat: a method for cell transplantation.

We examined the distribution of hippocampus-derived neural stem cells on the spinal cord surface for up to 3 weeks following injection through the fourth ventricle. The injected cells were disseminated as tiny spots on the pia mater of the spinal cord and proliferated into large cell-clusters. On both the dorsal and ventral side, cell clusters increased in number rapidly up to 5 days after injection and thereafter decreased gradually due to the coalition of neighbouring clusters. Concomitantly, individual cell clusters continuously increased in size, occupying almost 50% of the spinal cord surface. Cell attachment was usually found around blood vessels, along which cells invaded into the spinal cord. In the injured site, cells migrated into the lesion and were integrated into the spinal cord tissue, some of which had differentiated into astrocytes 1-2 weeks after injection. BrdU-uptake experiments demonstrated that the transplanted cells proliferated within the host cerebrospinal fluid. These results indicate that application of neural stem cells through the ventricle is an effective method to disseminate cells all over the spinal cord and that they can migrate and be integrated into the injured spinal cord.

In vivo infusions of exogenous growth factors into the fourth ventricle of the adult mouse brain increase the proliferation of neural progenitors around the fourth ventricle and the central canal of the spinal cord.

Stem cells isolated from the fourth ventricle and spinal cord form neurospheres in vitro in response to basic fibroblast growth factor (FGF2)+heparin (H) or epidermal growth factor (EGF)+FGF2 together. To determine whether these growth factor conditions are sufficient to induce stem cells within the fourth ventricle and spinal cord to proliferate and expand their progeny in vivo, we infused EGF and FGF2, alone or together, with or without H, into the fourth ventricle for 6 days via osmotic minipumps. Animals were injected with bromodeoxyuridine (BrdU) on days 4, 5 and 6 of infusion in order to label cells proliferating in response to the growth factors. Infusions of EGF+FGF2+H into the fourth ventricle resulted in the largest proliferative effect, a 10.8-fold increase in the number of BrdU+ cells around the fourth ventricle, and a 33.5-fold increase in the number of BrdU+ cells around the central canal of the spinal cord, as compared to vehicle infused controls. The majority of the cells were nestin+ after 6 days of infusion. Seven weeks post-infusion, 22 and 30% of the number of BrdU+ cells induced to proliferate after 6 days of EGF+FGF2+H infusions were still detected around the fourth ventricle and central canal of the spinal cord, respectively. Analysis of the fates of the remaining cells showed that a small percentage of BrdU+ cells around the fourth ventricle and in the white matter of the spinal cord differentiated into astrocytes and oligodendrocytes. BrdU+ neurons were not found in the brainstem or in the grey matter of the cervical spinal cord 7 weeks post-infusion. These results show that endogenous stem cells and progenitors around the fourth ventricle and central canal of the spinal cord proliferate in response to exogenously applied growth factors, but unlike in the lateral ventricle where they generate some new neurons, they only produce new astrocytes and oligodendrocytes at 7 weeks post-infusion.

Orexin-A depolarizes dissociated rat area postrema neurons through activation of a nonselective cationic conductance.

The area postrema (AP) is involved in the regulation of body fluid balance, feeding behavior, and cardiovascular function. Orexin (ORX)-A is a 33 aa peptide that regulates energy metabolism and sympathetic and cardiovascular actions. ORX immunoreactive axons and their varicose terminals have been found in AP. In this study, whole-cell, current- or voltage-clamp recordings were obtained from 108 dissociated rat AP neurons. The mean resting membrane potential of these neurons (n = 48) was -59.24 +/- 0.87 mV, the mean input resistance was 3.57 +/- 0.22 G(Omega), and the action potential amplitude of these cells was always >90 mV. Current-clamp studies showed bath application of ORX-A depolarized the majority of AP neurons tested (68.8%; 33 of 48), whereas small proportions of cells were either hyperpolarized (16.7%; 8 of 48) or unaffected (14.6%; 7 of 48). These depolarizing effects were found to be concentration dependent from 10(-8) to 10(-11) m. We then examined the contributions of specific ionic conductances to the ORX-A-induced excitation of AP neurons through whole-cell, voltage-clamp studies. Our results demonstrate that in contrast to previous studies on other neuronal populations, ORX-A did not affect net whole-cell potassium currents in AP neurons. Slow depolarizing voltage ramps, however, revealed that ORX-A enhanced a nonselective cationic conductance in AP neurons, effects which would explain the depolarizing effects of the peptide. These data demonstrate that AP neurons are directly influenced by ORX-A and suggest that ORX-A may exert its effects on the central control of feeding behavior and cardiovascular function through direct actions in AP.

Amylin and glucose co-activate area postrema neurons of the rat.

Glucose is an important metabolic factor controlling feeding behavior. There is evidence that physiologically relevant glucose sensors reside in the caudal hindbrain. The area postrema (AP) in particular, which has been characterized as a receptive site for the anorectic hormone amylin, may monitor blood glucose levels. To determine whether glucose and amylin co-activate the same subset of AP neurons, we performed extracellular single unit recordings from a rat AP slice preparation. In 53% of all AP neurons tested (n=32), the activity was positively correlated to the glucose concentration. Interestingly, there was a coincidental sensitivity (94%) of AP neurons to glucose and amylin, which exerted excitatory effects on these cells. We conclude that the co-sensitivity of AP neurons to glucose and amylin, both increasing in response to food intake, points to the AP as an important hindbrain center for the integration of the metabolic and hormonal control of nutrient intake.

Electrophysiological properties of the rat area postrema neurons displaying both the transient outward current and the hyperpolarization-activated inward current.

We found coexistence of the transient outward potassium current (I(TO)) and the hyperpolarization-activated inward current (I(H)) in 26 of 82 area postrema neurons tested using the whole-cell patch-clamp technique in rat brain slices. Cells displaying both the I(TO) and the I(H) typically showed "voltage sag" and "rebound potentials" in response to hyperpolarizing current injection and repetitive firing with strong adaptation was seen with depolarizing current injection. When cells were held at membrane potentials more negative than the resting level (e.g., -85mV), the afterhyperpolarization was enhanced. Voltage clamp recordings were performed to examine the characteristics of I(TO) and I(H) in and the contributions of these currents to the electroresponsiveness of area postrema cells. We show, in this study, the voltage-dependent properties of I(H) and I(TO), and how these currents modulate the intrinsic membrane properties of area postrema cells. We discuss the functional significance of the specific subset of area postrema neurons whose cells have both I(H) and I(TO) channels.

Bcl-2 expression in thalamus, brainstem, cerebellum and visual cortex of adult primate.

Due to the functional importance of Bcl-2, which acts as an anti-apoptotic protein that also affects neural differentiation and adult neurogenesis, we undertook a detailed immunohistochemical study of the distribution of this protein in the brain of squirrel monkeys. The present study describes findings obtained at thalamic, brainstem, cerebellum and visual cortex levels, and the data are compared with our previous results gathered in the same species. At thalamic level, Bcl-2-positive neurons occur in anterior, rostral intralaminar, midline and lateral habenular nuclei. The protein is also expressed in several structures associated with the ventricular system, including the subventricular zone (SVZ), the subcommissural organ, and the periventricular grey at rostral and caudal tips of the fourth ventricle. At brainstem and cerebellar levels, Bcl-2-positive neurons occur in the dorsal raphe nucleus, inferior olivary complex, and in molecular and granular layers of the cerebellum. Finally, neurons of layer IV of the striate cortex display a very strong Bcl-2 immunoreactivity that contrasts with the poor labeling of neurons in adjacent parastriate and peristriate cortices. These finding suggests that Bcl-2 plays a role in the plasticity and structural maintenance of various structures in the primate brain and indicate that the mitotically active SVZ might be more extended along the rostrocaudal axis in primates than in rodents.

Two distinct types of transient outward currents in area postrema neurons in rat brain slices.

We investigated the electrophysiological properties of the area postrema neurons in acutely prepared rat brain slices using the whole-cell patch-clamp technique. Two different types of transient outward potassium current (I(to)), fast and slow, were found in the area postrema. Both the decay time constant and rise time were significantly faster in the fast I(to) than in the slow I(to). Both current-clamp and voltage-clamp recordings revealed that the activation of fast and slow I(to) contributes to generation of the different spiking patterns, late spiking and interrupted spiking, respectively. The activation and inactivation of both I(to) were strongly voltage-dependent. Curve fitting by the Boltzmann equation revealed no significant difference in the activation and inactivation curves for each I(to) except that the slope factor of inactivation was larger for fast I(to). Both I(to) were suppressed dose-dependently by application of 4-aminopyridine. Each spiking pattern was enhanced when cells were held at a more hyperpolarized membrane potential, i.e. a longer latency of the first spike or longer interspike interval between the first and second spikes. The voltage-dependent modulation of the spiking pattern was consistent with the voltage-dependent activation of I(to). The present study shows significant subdivisions of the area postrema neurons distinguished by a difference in the kinetics of I(to) and spiking patterns. We discuss the role of I(to) as the ionic current underlying neuronal excitability.