Hideto Kaba

Identification of two highly homologous presynaptic proteins distinctly localized at the dendritic and somatic synapses

Through screening of a murine brain cDNA library, we have isolated two brain specific cDNAs encoding highly homologous proteins, named 921‐L and 921‐S, comprised of 134 amino acids with 80% identity. Immunohistological study with the mAbs raised against the bacterially expressed 921 proteins showed that 921‐L protein is distributed at the dendritic region and 921‐S at the neuronal somatic surface. Immuno‐electron microscopic study revealed that both 921 proteins are localized at the presynaptic terminal, indicating that the 921 proteins are differentially expressed at the dendritic and somatic presynapses.

The olfactory bulb : A critical site of action for oxytocin in the induction of maternal behaviour in the rat

Expanding on research showing that oxytocin originating in the hypothalamic paraventricular nucleus acts to decrease olfactory processing at the level of the olfactory bulb, we explored the importance of oxytocin acting on the olfactory bulb for the onset of maternal behaviour in Wistar rats. Experiment I was designed to test whether spontaneous maternal behaviour following natural delivery is blocked by bilateral infusions of a low dose (5 fmol) of the oxytocin antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]ornithine-vasotocin into the olfactory bulb immediately after the delivery of the first pup and again just before a test for maternal behaviour. Intrabulbar infusions of the antagonist markedly delayed the occurrence of all components (retrieval, licking, nest building, crouching) of maternal behaviour, whereas intracerebroventricular infusions of the antagonist were without effect on any component as compared with intrabulbar infusions of saline. Experiment 2 was undertaken to determine whether infusions of oxytocin into the bulb induce a rapid onset of maternal behaviour in virgin rats. Forty-eight hours before pup presentation virgins were ovariectomized and treated with oestradiol benzoate. Immediately before pup presentation a low dose (20 pmol) of oxytocin or saline was infused bilaterally into the bulb or lateral ventricle. Intrabulbar infusions of oxytocin induced full maternal behaviour in half of the animals tested within 2 h of pup exposure, in contrast to the ineffectiveness of intracerebroventricular infusions of oxytocin and intrabulbar infusions of saline. These results suggest that the olfactory bulb is a critical site where oxytocin acts to induce a rapid onset of maternal behaviour.

Synaptic mechanisms of olfactory recognition memory

The complexity and inaccessibility of the mammalian brain prevent the localization and description of memory traces and the definition of the processes that produce memories. The model reviewed here is the olfactory recognition memory formed to male pheromones by a female mouse at mating. The memory trace has been localized to the reciprocal dendrodendritic synapse between mitral cells and granule cells in the accessory olfactory bulb. An increase in noradrenaline after mating reduces inhibitory transmission of gamma-aminobutyric acid (GABA) from the granule cells to mitral cells and induces an olfactory memory of pheromones present at mating. Recent work has shown that the activation of mGluR2, a metabotropic glutamate receptor, localized at granule cell dendrites suppresses the GABA inhibition of the mitral cells and permits the formation of a specific olfactory memory that faithfully reflects the memory formed at mating. This simple olfactory memory may provide an excellent model system with which to investigate the molecular mechanisms of the synaptic plasticity involved in learning and memory.

Electrophysiological evidence that circulating angiotensin II sensitive neurons in the subfornical organ alter the activity of hypothalamic paraventricular neurohypophyseal neurons in the rat

Thirteen neurons in the subfornical organ (SFO) were antidromically activated by electrical stimulation of the paraventricular nucleus (PVN) in the rat. The activity of these identified SFO neurons was excited by intravenous injection of angiotensin II (AII). Electrical stimulation of the SFO produced orthodromic excitation (40%) and inhibition (40%) of the activity of putative vasopressin (VP)-secreting PVN neurons. These results suggest that circulating AII sensitive SFO neurons with efferent projections to the PVN have both excitatory and inhibitory influences on the activity of putative VP-secreting neurons in the PVN.

Reduced hippocampal LTP in mice lacking a presynaptic protein: complexin II.

The SNAP receptor (SNARE) complex is a core complex specialized for synaptic vesicle exocytosis, and the binding of SNAPs to the complex is an essential step for neurotransmitter release. Complexin I and II have been identified as SNARE‐complex‐associated proteins. Importantly, complexins compete with α‐SNAP for binding to the complex, suggesting that complexins may modulate neurotransmitter release process. To examine this possibility and to understand the physiological function of complexins, we generated complexin II knockout mice. The complexin‐II‐deficient mice (–/–) were viable and fertile, and appeared normal. Electrophysiological recordings in the mutant hippocampus showed that ordinary synaptic transmission and paired‐pulse facilitation, a form of short‐term synaptic plasticity, were normal. However, long‐term potentiation (LTP) in both CA1 and CA3 regions was impaired, suggesting that complexin II may not be essential for synaptic vesicle exocytosis, but it does have a role in the establishment of hippocampal LTP.

The action of oxytocin originating in the hypothalamic paraventricular nucleus on mitral and granule cells in the rat main olfactory bulb.

The effects of electrical stimulation of the hypothalamus paraventricular nucleus on the spontaneous firing of mitral and granule cells in the main olfactory bulb were examined in ovariectomized female rats under urethane anaesthesia. High-frequency stimulation (0.5-1.0 mA, 10-20 pulses at 100 Hz) of the paraventricular nucleus produced inhibitory responses in 80% of mitral cells tested and excitatory responses in 74% of granule cells tested, with latencies ranging from 2 to 150 s. Both responses were blocked by infusions into the olfactory bulb of [d(CH2)5, Tyr(Me)2]ornithine-vasotocin (10 pmol), an oxytocin antagonist, and mimicked by intracerebroventricular infusions (0.2 or 0.4 nmol) or microiontophoretic applications of oxytocin but not by intracerebroventricular infusions of vasopressin (1 or 2 nmol). Infusions of 0.5% lignocaine, a local anaesthetic, into either the medial olfactory tract or the medial forebrain bundle failed to block the responses of mitral and granule cells to the stimulation. Unilateral transections at various levels between the bulb and the paraventricular nucleus also failed to block the responses. There were cases in which significant responses of mitral and granule cells to the stimulation required 60 or more pulses after the lignocaine infusions or transections, however. These results suggest that oxytocin originating in the hypothalamic paraventricular nucleus reaches the olfactory bulb following its release partly into the cerebrospinal fluid and acts to decrease olfactory processing.

Synaptic plasticity in olfactory memory formation in female mice

FEMALE mice develop a long-lasting olfactory recognition memory of a partner male at the first relay in the vomeronasal system. In this study the synaptic plasticity relevant to this phenomenon was examined at reciprocal dendrodendritic synapses in the accessory olfactory bulb of female mice by electron microscopy. The size of asymmetrical excitatory synapses (mitral/tufted to granule cells) of the reciprocal synapses was significantly larger in the group of female mice which were subjected to a treatment intended to induce olfactory memory formation than in the control group. It is suggested that olfactory memory formation is associated with a conformational change at the level of synaptic structure of the accessory olfactory bulb.

Subfornical organ neurons with efferent projections to the hypothalamic paraventricular nucleus: an electrophysiological study in the rat

Seventeen neurons in the subfornical organ (SFO) were antidromically activated by electrical stimulation of the paraventricular nucleus (PVN) in the rat. The activity of all identified SFO neurons was excited by microiontophoretically (MIPh) applied angiotensin II (AII) and the effect of AII was blocked by MIPh-applied saralasin (Sar), an AII antagonist, but not by atropine (Atr), a muscarinic antagonist. In these identified SFO neurons, 9 were also excited and 8 were not affected by MIPh-applied acetylcholine (ACh) and the effect of ACh was attenuated by not only MIPh-applied Atr but also by Sar. These results suggest that there are specific AII- and both AII- and ACh-sensitive types of SFO neurons with efferent projections to the PVN.

Inputs from the A1 noradrenergic region to hypothalamic paraventricular neurons in the rat

Electrical stimulation of the rat A1 noradrenergic region produced excitation (77%) of the activity of putative vasopressin (VP)-secreting neurons in the paraventricular nucleus (PVN) and produced excitation (4%), inhibition (26%) and excitation-inhibition (11%) of the activity of PVN neurons that were not antidromically identified by neurohypophysial stimulation. The excitatory response of putative VP-secreting neurons was blocked by microiontophoretically applied phentolamine, an alpha-adrenoceptor antagonist, but not by timolol, a beta-adrenoceptor antagonist. The inhibitory response of unidentified PVN neurons, on the other hand, was blocked by timolol, but not by phentolamine.

Activation of the cyclic AMP response element-binding protein signaling pathway in the olfactory bulb is required for the acquisition of olfactory aversive learning in young rats.

Long-term memory formation requires both gene expression and protein synthesis. Phosphorylation of the transcription factor cyclic AMP response element-binding protein (CREB) is thought to be important in processes underlying long-term memory. To clarify the role of CREB in olfactory aversive learning in young rats, we carried out behavioral pharmacology and Western blot analyses. On postnatal day 11, oligodeoxynucleotides were infused directly into the bilateral olfactory bulbs through cannulae implanted prior to training in a classical conditioning paradigm with citral odor and foot shock. On the following day the odor preference test was performed. After training, saline-infused animals spent significantly shorter time over the citral odor zone. Infusion of CREB antisense oligodeoxynucleotides 6 h before or during training, however, prevented olfactory aversive learning without affecting memory retention 1 h after training. CREB scrambled oligodeoxynucleotides infusions had no effect on olfactory learning. When infused 6 h after training, none of oligodeoxynucleotides had an effect on time spent over the odor zone. Using Western blotting, we analyzed CREB in nuclear extracts obtained from the young rats after training. Marked increases in phosphorylated CREB were sustained from 10 to 360 min after the odor-shock pairing in animals which were subjected to both, in comparison with levels 30 min in animals which were subjected to odor only or no stimulation. Total CREB levels showed no differences among groups. Infusion of CREB antisense oligodeoxynucleotides significantly reduced the expression of phosphorylated and total CREBs in the olfactory bulb. These results show that the synthesis and phosphorylation of CREB are required for the acquisition of olfactory aversive learning in young rats, and that this requirement for the CREB signaling pathway has a critical time window.