Maternally inherited peptides as strain-specific chemosignals.

Most mammals rely on chemosensory cues for individual recognition, which is essential to many aspects of social behavior, such as maternal bonding, mate recognition, and inbreeding avoidance. Both volatile molecules and nonvolatile peptides secreted by individual conspecifics are detected by olfactory sensory neurons in the olfactory epithelium and the vomeronasal organ. The pertinent cues used for individual recognition remain largely unidentified. Here we show that nonformylated, but not N-formylated, mitochondrially encoded peptides-that is, the nine N-terminal amino acids of NADH dehydrogenases 1 and 2-can be used to convey strain-specific information among individual mice. We demonstrate that these nonformylated peptides are sufficient to induce a strain-selective pregnancy block. We also observed that the pregnancy block by an unfamiliar peptide derived from a male of a different strain was prevented by a memory formed at the time of mating with that male. Our findings also demonstrate that pregnancy-blocking chemosignals in the urine are maternally inherited, as evidenced by the production of reciprocal sons from two inbred strains and our test of their urine's ability to block pregnancy. We propose that this link between polymorphic mitochondrial peptides and individual recognition provides the molecular means to communicate an individual's maternal lineage and strain.

Olfactory Stimulation with Japanese Soy Sauce Improves Upper Limb Performance.

BACKGROUND: We have observed changes in body reactions during cooking, which is one of the treatment modalities used in occupational therapy. The perception of food-related odors during cooking may have behavioral effects on human activities through the activation of appetitive motivation. OBJECTIVES: We investigated whether odor components contained in seasonings could facilitate the human motor system and the specificity of this effect. METHODS: The subjects were 72 healthy adults, randomly assigned to a water exposure group, a phenylethyl alcohol (PEA, pleasant rose-like odor) exposure group, and a Japanese soy sauce (Koikuchi Shoyu) exposure group (n = 24 each). The subjects' olfactory sense was stimulated by their sniffing of three different test tubes containing 5 ml of water, PEA, or Japanese soy sauce for 20 sec while they were seated. The modified Functional Reach Test (mFRT), which mimics a functional activity that is required in daily living and assesses a reliable measure of sitting balance, was performed prior to and immediately after the sniffing. RESULTS: Sniffing the soy sauce increased the subjects' mFRT scores. This facilitation effect was odorant-specific and was absent when the subjects were presented with water or PEA. CONCLUSIONS: Cooking interventions are aimed at improving tool-handling skills such as using knives and chopsticks. The results indicate that treatment interventions using odors of seasonings would be effective for improving subjects' physical functions.

Representations of microgeometric tactile information during object recognition.

Object recognition through tactile perception involves two elements: the shape of the object (macrogeometric properties) and the material of the object (microgeometric properties). Here we sought to determine the characteristics of microgeometric tactile representations regarding object recognition through tactile perception. Participants were directed to recognize objects with different surface materials using either tactile information or visual information. With a quantitative analysis of the cognitive process regarding object recognition, Experiment 1 confirmed the same eight concepts (composed of rules defining distinct cognitive processes) commonly generated in both tactile and visual perceptions to accomplish the task, although an additional concept was generated during the visual task. Experiment 2 focused only on tactile perception. Three tactile objects with different surface materials (plastic, cloth and sandpaper) were used for the object recognition task. The participants answered a questionnaire regarding the process leading to their answers (which was designed based on the results obtained in Experiment 1) and to provide ratings on the vividness, familiarity and affective valence. We used these experimental data to investigate whether changes in material attributes (tactile information) change the characteristics of tactile representation. The observation showed that differences in tactile information resulted in differences in cognitive processes, vividness, familiarity and emotionality. These two experiments collectively indicated that microgeometric tactile information contributes to object recognition by recruiting various cognitive processes including episodic memory and emotion, similar to the case of object recognition by visual information.

α2-Adrenergic receptor activation promotes long-term potentiation at excitatory synapses in the mouse accessory olfactory bulb.

The formation of mate recognition memory in mice is associated with neural changes at the reciprocal dendrodendritic synapses between glutamatergic mitral cell (MC) projection neurons and GABAergic granule cell (GC) interneurons in the accessory olfactory bulb (AOB). Although noradrenaline (NA) plays a critical role in the formation of the memory, the mechanism by which it exerts this effect remains unclear. Here we used extracellular field potential and whole-cell patch-clamp recordings to assess the actions of bath-applied NA (10 µM) on the glutamatergic transmission and its plasticity at the MC-to-GC synapse in the AOB. Stimulation (400 stimuli) of MC axons at 10 Hz but not at 100 Hz effectively induced N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP), which exhibited reversibility. NA paired with subthreshold 10-Hz stimulation (200 stimuli) facilitated the induction of NMDA receptor-dependent LTP via the activation of α2-adrenergic receptors (ARs). We next examined how NA, acting at α2-ARs, facilitates LTP induction. In terms of acute actions, NA suppressed GC excitatory postsynaptic current (EPSC) responses to single pulse stimulation of MC axons by reducing glutamate release from MCs via G-protein coupled inhibition of calcium channels. Consequently, NA reduced recurrent inhibition of MCs, resulting in the enhancement of evoked EPSCs and spike fidelity in GCs during the 10-Hz stimulation used to induce LTP. These results suggest that NA, acting at α2-ARs, facilitates the induction of NMDA receptor-dependent LTP at the MC-to-GC synapse by shifting its threshold through disinhibition of MCs.

Defective imitation of finger configurations in patients with damage in the right or left hemispheres: An integration disorder of visual and somatosensory information?

To explore the mechanism underlying the imitation of finger gestures, we devised a simple imitation task in which the patients were instructed to replicate finger configurations in two conditions: one in which they could see their hand (visual feedback: VF) and one in which they could not see their hand (non-visual feedback: NVF). Patients with left brain damage (LBD) or right brain damage (RBD), respectively, were categorized into two groups based on their scores on the imitation task in the NVF condition: the impaired imitation groups (I-LBD and I-RBD) who failed two or more of the five patterns and the control groups (C-LBD and C-RBD) who made one or no errors. We also measured the movement-production times for imitation. The I-RBD group performed significantly worse than the C-RBD group even in the VF condition. In contrast, the I-LBD group was selectively impaired in the NVF condition. The I-LBD group performed the imitations at a significantly slower rate than the C-LBD group in both the VF and NVF conditions. These results suggest that impaired imitation in patients with LBD is partly due to an abnormal integration of visual and somatosensory information based on the task specificity of the NVF condition.

Perception of active head rotation in patients with severe left unilateral spatial neglect.

Unilateral spatial neglect is a common neurological syndrome following predominantly right hemisphere damage, and is characterized by a failure to perceive and report stimuli in the contralesional side of space. To test the reference shift hypothesis that contralesional spatial neglect in right-brain-damaged patients is attributed to a rightward deviation of the egocentric reference frame, we measured the final angular position to which controls and left-side neglect patients actively turned their head toward the left in response to a verbal instruction given from each of three locations-right, left, and front-in two conditions, with and without visual feedback. When neglect patients were asked to "look straight ahead", they deviated about 30° toward the right in the eyes-open condition. However, the rightward deviation was markedly reduced in the eyes-closed condition. Regardless of visual feedback, there was no significant difference between controls and neglect patients in the final angular position of active head rotation when the verbal instruction came from the subject's left or front side; however, the final angular position was significantly smaller in the neglect patients than in the controls when the verbal instruction was given from the right. These results support the contention that cervico-vestibular stimulation during active head rotation restores spatial remapping and sensori-motor correlations and so improves neglect without affecting the position of the egocentric reference; however, once left-side neglect patients respond to verbal instruction from the right side, they are unable to disengage attention from the hemispace, and the performance of head rotation is disturbed.

Tunicamycin impairs olfactory learning and synaptic plasticity in the olfactory bulb.

Tunicamycin (TM) induces endoplasmic reticulum (ER) stress and inhibits N-glycosylation in cells. ER stress is associated with neuronal death in neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease, and most patients complain of the impairment of olfactory recognition. Here we examined the effects of TM on aversive olfactory learning and the underlying synaptic plasticity in the main olfactory bulb (MOB). Behavioral experiments demonstrated that the intrabulbar infusion of TM disabled aversive olfactory learning without affecting short-term memory. Histological analyses revealed that TM infusion upregulated C/EBP homologous protein (CHOP), a marker of ER stress, in the mitral and granule cell layers of MOB. Electrophysiological data indicated that TM inhibited tetanus-induced long-term potentiation (LTP) at the dendrodendritic excitatory synapse from mitral to granule cells. A low dose of TM (250nM) abolished the late phase of LTP, and a high dose (1µM) inhibited the early and late phases of LTP. Further, high-dose, but not low-dose, TM reduced the paired-pulse facilitation ratio, suggesting that the inhibitory effects of TM on LTP are partially mediated through the presynaptic machinery. Thus, our results support the hypothesis that TM-induced ER stress impairs olfactory learning by inhibiting synaptic plasticity via presynaptic and postsynaptic mechanisms in MOB.

Activation of arginine vasopressin receptor 1a facilitates the induction of long-term potentiation in the accessory olfactory bulb of male mice.

Olfaction plays an important role in social recognition in most mammals. Central arginine vasopressin (AVP) plays a role in this olfaction-based recognition. The high level of expression of AVP receptors in the accessory olfactory bulb (AOB) at the first relay of the vomeronasal system highlights the importance of AVP signaling at this stage. We therefore analyzed the effects of AVP on the synaptic plasticity of glutamatergic transmission from mitral cells to granule cells in AOB slices from male mice. To monitor the strength of the glutamatergic transmission, we measured the maximal initial slope of the lateral olfactory tract-evoked field potential, which represents the granule cell response to mitral cell activation. AVP paired with 100-Hz stimulation that only produced short-term potentiation enhanced the induction of long-term potentiation (LTP) in a dose-dependent manner. AVP-paired LTP was blocked by the selective AVP receptor 1a (AVPR1a) antagonist, d(CH2)5[Tyr(Me)2]AVP (Manning compound), but not by the AVPR1b antagonist SSR149415, and it was mimicked by the selective AVPR1a agonist [Phe2, Ile3, Orn8]-vasopressin. We further examined the effect of AVP on the reciprocal transmission between mitral and granule cells by stimulating a mitral cell and recording the evoked inhibitory postsynaptic currents (IPSCs) from the same cell using conventional whole-cell patch-clamp techniques. AVP reduced the reciprocal IPSCs triggered by endogenous glutamate release from the excited mitral cell. These results suggest that AVP promotes the induction of LTP at the mitral-to-granule cell synapse via the activation of AVPR1a through an as-yet-to-be-determined mechanism in the AOB of male mice.

Comparison of timing and force control of foot tapping between elderly and young subjects.

[Purpose] To examine the ability of young and elderly individuals to control the timing and force of periodic sequential foot tapping. [Subjects and Methods] Participants were 10 young (age, 22.1 ± 4.3 years) and 10 elderly individuals (74.8 ± 6.7 years) who were healthy and active. The foot tapping task consisted of practice (stimulus-synchronized tapping with visual feedback) and recall trials (self-paced tapping without visual feedback), periodically performed in this order, at 500-, 1,000-, and 2,000-ms target interstimulus-onset intervals, with a target force of 20% maximum voluntary contraction of the ankle plantar-flexor muscle. [Results] The coefficients of variation of force and intertap interval, used for quantifying the steadiness of the trials, were significantly greater in the elderly than in the young individuals. At the 500-ms interstimulus-onset interval, age-related effects were observed on the normalized mean absolute error of force, which was used to quantify the accuracy of the trials. The coefficients of variation of intertap interval for elderly individuals were significantly greater in the practice than in the recall trials at the 500- and 1,000-ms interstimulus-onset intervals. [Conclusion] The elderly individuals exhibited greater force and timing variability than the young individuals and showed impaired visuomotor processing during foot tapping sequences.

Evaluation of "Open Essence" odor-identification test card by application to healthy volunteers.

OBJECTIVE: In the United States and European countries, the University of Pennsylvania Smell Identification Test (UPSIT), the Quick Smell Identification Test (Q-SIT), a shorter version of the UPSIT, and "Sniffin' Sticks" are widely used for testing olfaction. The odor stick identification test for Japanese (OSIT-J) has been manufactured in Japan. This was followed by the development of "Open Essence," consisting of cards that make use of sealed odorants. This study examined the usefulness of Open Essence in young volunteers with normal olfaction. METHOD: The participants were 176 medical students in their clinical clerkships at the Department of Otolaryngology, Kochi Medical School Hospital. There were 111 males and 65 females, with a median age of 24 years (range 22-42 years). The olfactory function of all participants was confirmed as normal by screening with T&T olfactometry. Before beginning Open Essence, the subjects were asked to answer a questionnaire on their olfaction and habits. RESULTS: Females had significantly better smell identification than males. Of the 12 odorants, "perfume" and "cooking gas" were difficult to identify, because perfume involves different imagery and the compounds that give the smell of gas are readily volatilized. In contrast, all participants recognized "curry" and "menthol," probably because they are received as both somatosensory stimuli and smell. CONCLUSIONS: These results suggest that, with improvement, Open Essence might be used for testing olfaction in Japanese subjects.

Regulation of synaptic currents by mGluR2 at reciprocal synapses in the mouse accessory olfactory bulb.

The throughput of information from the accessory olfactory bulb (AOB) to downstream structures is controlled by reciprocal dendrodendritic inhibition of mitral cells by granule cells. Given the high expression levels of mGluR2, a metabotropic glutamate receptor, in the AOB and the fact that the activation of mGluR2 permits the formation of a specific olfactory memory, we reasoned that mGluR2 might play an important role in regulating dendrodendritic inhibition. To test this hypothesis, we examined the effects of pharmacological and genetic manipulations of mGluR2 on synaptic responses measured from mitral or granule cells in slice preparations from 23- to 36-day-old Balb/c mice. To evoke dendrodendritic inhibition, a depolarizing voltage step from -70 to 0 mV or a threshold current stimulus adjusted to elicit action potential(s) was applied to a mitral cell using either a nystatin-perforated or conventional whole-cell configuration. We found that an agonist for group II metabotropic glutamate receptors (mGluR2/mGluR3), DCG-IV [(2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine], suppressed, whereas the mGluR2/mGluR3 antagonist LY341495 [(αS)-α-amino-α-[(1S,2S)-2-carboxycyclopropyl]-9H-xanthine-9-propanoic acid] enhanced dendrodendritic inhibition. Genetic ablation of mGluR2 markedly impaired the effects of DCG-IV and LY341495 on dendrodendritic inhibition. DCG-IV reduced both the frequency and the amplitude of spontaneous miniature excitatory postsynaptic currents recorded from granule cells. Additionally, DCG-IV inhibited high-voltage-activated calcium currents in both mitral and granule cells. These results suggest that mGluR2 reduces dendrodendritic inhibition by inhibiting synaptic transmission between mitral cells and granule cells in the AOB.

Accessory olfactory bulb neurons are required for maintenance but not induction of V2R vomeronasal receptor gene expression in vitro.

Many mammals detect pheromones by a sensory organ, the vomeronasal organ (VNO). In a previous study using immunoblot and immunocytochemical analyses, we reported that cocultures of VNOs with accessory olfactory bulb (AOB) neurons resulted in the maturation of vomeronasal sensory neurons (VSNs) and a greater expression of V2R family vomeronasal receptors than cultures with VNO alone. To further characterize the V2R expression, we here investigated the time course of the expression of V2R mRNA in the presence or absence of AOB neurons using RT-PCR analysis. The expression of V2R mRNA was already detectable not only in the VNO cocultured with AOB neurons for 3 days in coculture but also in the VNO cultured alone for the same number of days. However, the expression of V2R mRNA in the VNO cultured alone was remarkably decreased during the additional culture period, although that in the cocultured VNO showed sustained expression. Moreover, the application of 2 µM TTX to the cocultured VNO resulted in a marked decrease in the V2R mRNA expression to a level equal to that in the VNO cultured alone for 14 days in coculture. Our previous working hypothesis was that the expression of V2Rs in VSNs was induced by interacting with AOB neurons. However, the present results suggest that the receptor expression in VSNs is independent of the interaction with AOB neurons in the early developmental stage, but is maintained by the active interaction with AOB neurons.

Ameliorative effects of Eriobotrya japonica seed extract on cellular aging in cultured rat fibroblasts.

To investigate the effects of Eriobotrya japonica seed extract (ESE) on cellular aging, intracellular calcium homeostasis in young and senescent cells was analyzed using a rat fibroblast culture as an in vitro model system and a calcium imaging technique. The application of bradykinin (BK) transiently elicited intracellular calcium ion (Ca(2+)) increased in most of the young fibroblasts, whereas these responses were scarcely observed or were significantly attenuated in senescent cells. However, the long-term treatment of senescent cells with ESE (for 7 days) dose-dependently increased the amplitude of BK-induced responses and the percentage of BK-responding cells. In particular, most senescent cells could respond to BK with long-term treatment with ESE (1.0% or 2.0%), an effect that reinstated the percentage of BK-responding cells to the same level as that in young cells. The effects of ESE on amplitude or percentage of responding cells were not observed in young cells. Moreover, the time to half decay, which was significantly longer in senescent cells than that in young cells, was shortened in senescent cells with long-term treatment with ESE. These results suggest that treatment with an adequate concentration of ESE renders BK-induced Ca(2+) dynamics in senescent cells similar to those in young cells. Therefore, ESE can retard and/or protect against cellular aging and may be useful for elucidating the antiaging processes.

Muscarinic receptor type 1 (M1) stimulation, probably through KCNQ/Kv7 channel closure, increases spontaneous GABA release at the dendrodendritic synapse in the mouse accessory olfactory bulb.

Cholinergic modulation of spontaneous GABAergic currents (mIPSC) was studied using whole-cell patch methods in mouse accessory olfactory bulb slices. Carbachol (above 100 microM) administration produced an increase in the mIPSC frequency in mitral cells, but did not affect the responses of mitral cells to GABA. The carbachol effect persisted in the presence of combined ionotropic and metabotropic glutamatergic receptor antagonists. The carbachol effect was reduced by the muscarinic receptor type-1 and -4 (M1 and M4) antagonist pirenzepine (10 microM), but not by the M2 and M4 antagonist himbacine (10 microM). The KCNQ/Kv7 potassium channel openers retigabine (80 microM) and diclofenac (300 microM) blocked the carbachol action, while the KCNQ potassium channel blocker XE-911 (20 microM) increased the mIPSC frequency. XE-911's action persisted in the presence of glutamate receptor blockers. In the presence of carbachol, mIPSCs were abolished by Ni (200 microM), while being insensitive to the calcium channel blocker nimodipine (30 microM), suggesting a role for R-type calcium channels in the GABA release. These results suggest that carbachol closed KCNQ channels by stimulating M1 receptors on granule cell dendrites, and the resulting depolarized and unstable membrane promoted calcium influx, thus increasing the GABA release. The possible role of acetylcholine in facilitating formation of a pheromone memory in mice is also discussed.

The effect of vapor of propylene glycol on rats.

Propylene glycol (PG) is commonly used as a solvent for odorous chemicals employed in studies of the olfactory system because PG has been considered to be odorless for humans and other animals. However, if laboratory rats can detect the vapor of PG and if exposure to this influences behaviors, such effects might confound data obtained from experiments exposing conscious rats to odorants dissolved in PG. Therefore, we examined this issue using differences in the acoustic startle reflex (ASR) as an index. We also conducted a habituation/dishabituation test to assess the ability of rats to detect the vapor of PG. In addition, we observed Ca(2+) responses of vomeronasal neurons (VNs) in rats exposed to PG using the confocal Ca(2+)-imaging approach. Pure PG vapor significantly enhanced the ASR at a dose of 1 x 10(-4) M, which was much lower than the dose for efficiently detecting. In Ca(2+) imaging, VNs were activated by PG at a dose of 1 x 10(-4) M or lower. These results suggest that PG vapor acts as an aversive stimulus to rats at very low doses, even lower than those required for its detection, indicating that we should consider such effect of PG when it is employed as a solvent for odorants in studies using conscious rats. In addition, our study suggests that some non-pheromonal volatile odorants might affect animal behaviors via the vomeronasal system.

Oxytocin facilitates the induction of long-term potentiation in the accessory olfactory bulb.

When female mice are mated, they form a memory to the pheromonal signal of their male partner. Several lines of evidence indicate that the neural changes underlying this memory occur in the accessory olfactory bulb (AOB) at the first stage of the vomeronasal system. The formation of this memory depends on the mating-induced release of noradrenaline in the AOB. In addition to noradrenaline, the neuropeptide oxytocin (OT) is also released within the central nervous system during mating. Because OT has been implicated in social memory and its receptors are expressed in the AOB, we hypothesized that OT might promote the strength of synaptic transmission from mitral to granule cells in the AOB. To test this hypothesis, we analyzed the lateral olfactory tract-evoked field potential that represents the granule cell response to mitral cell activation and its plasticity in parasagittal slices of the AOB. Of the 10-, 20-, 50-, and 100-Hz stimulations tested, the 100-Hz stimulation was optimal for inducing long-term potentiation (LTP). OT paired with 100-Hz stimulation that only produced short-term potentiation enhanced LTP induction in a dose-dependent manner. OT-paired LTP was blocked by both the selective OT antagonist desGly-NH(2),d(CH(2))(5)[Tyr(Me)(2),Thr(4)]-ornithine vasotocin and the N-methyl-d-aspartate (NMDA) receptor antagonist dl-2-amino-5-phosphonovaleric acid. These results indicate that OT can function as a gate to modulate the establishment of NMDA receptor-dependent LTP at the mitral-to-granule cell synapse in the AOB.

Target regulation of V2R expression and functional maturation in vomeronasal sensory neurons in vitro.

Vomeronasal receptors from the V1R and V2R gene families mediate the detection of chemical stimuli such as pheromones via the vomeronasal organ (VNO). The differential expression of vomeronasal receptors might contribute in part to a variety of pheromonal effects, which are different sexually, developmentally and even individually. However, little is known about the mechanisms controlling vomeronasal receptor expression. Cultured vomeronasal sensory neurons (VSNs) bear phenotypic resemblance to the intact VNO but they remain immature. Because indices of VSN maturation are increased by coculture with the target cells for VSNs, accessory olfactory bulb (AOB) neurons, AOB neurons may regulate vomeronasal receptor expression and functional maturation in VSNs. To test this hypothesis, we examined the expression of V2R-type vomeronasal receptors (VR1 and VR4) and chemosensory responsiveness in VNOs cocultured with AOB neurons. Immunoblot and immunocytochemical analysis revealed that the coculture of VNOs with AOB neurons resulted in a greater expression of VR1 and VR4 after 10 days than VNOs cultured alone. Moreover, calcium imaging analysis showed that cocultured VNOs responded to urine components applied iontophoretically into their cavities with a time course similar to the V2R expression, in contrast to singly cultured VNOs that displayed no response. These results demonstrate that AOB neurons induce the expression of vomeronasal receptors in VSNs, allowing them to function.

Effects of olfactory stimulation with isovaleric acid on brain activation in informed and naïve conditions: a functional MRI study.

OBJECTIVE: To investigate the differences in regions of brain activation in response to olfactory stimulation by functional magnetic resonance imaging in conditions of prior warning of an odor and without. METHODS: Participants were 17 normal right-handed volunteers; 8 participants received prior warning of the odor (informed condition) and 9 participants were not pre-warned (naïve condition). The odorant used was isovaleric acid. RESULTS: In the informed condition with prior warning, activation was observed in the putamen extending to the insula, amygdala, and inferior frontal gyrus, and there was instant reification of the odor, while in the naïve condition without prior warning, activation was observed in the anterior cingulate cortex, entorhinal cortex, putamen and inferior frontal gyrus, and recognition of the odor was difficult. CONCLUSIONS: These results suggest that the condition prior to olfactory stimulation, i.e., with or without prior warning, can affect recognition and regions of brain activation in response to olfactory stimulation using isovaleric acid. Differences in recognition and regions of brain activation between both conditions could be associated with response latencies, or degree of attention, expectation and/or concentration.

Differences in development and cellular composition between neuronal cultures of rat accessory and main olfactory bulbs.

We previously established a primary culture system of the accessory olfactory bulb (AOB) to investigate the functional roles of individual types of neuron in pheromonal signal processing. However, the detailed characteristics of cultured AOB neurons were not yet apparent. In the present study, we address the cytological aspects of cultured AOB neurons using immunocytochemical staining methods. Cultured AOB neurons were compared with cultured main olfactory bulb (MOB) neurons in neuronal composition, maturational time course, and cell size. The number of total neurons, measured by microtubule-associated protein 2 (MAP2) immunostaining, progressively decreased, and glutamic acid decarboxylase positive (GAD+) interneurons were scarcely changed in their number in both AOB and MOB cultures over the culture periods. In contrast, the number of tyrosine hydroxylase positive (TH+) neurons in AOB cultures showed a slight, but significant, increase over time in culture, while those in MOB cultures remarkably decreased. The numbers of total neurons and GAD+ neurons were significantly greater in AOB cultures than in MOB cultures at all investigated time points. However, the numbers of TH+ neurons were lower at 7 days in vitro (DIV) and greater at 21 DIV in AOB cultures than in MOB cultures. The somatic sizes of all types of neurons at 14 DIV were significantly larger in AOB cultures than in MOB cultures. Furthermore, the frequency distributions of somatic sizes of total, GAD+, and TH+ neurons were significantly different between AOB and MOB cultures. These subtle differences in vitro may reflect in vivo differences between the AOB and MOB.