Dai Mitsushima

Effects of pulsatile infusion of the GABA(A) receptor blocker bicuculline on the onset of puberty in female rhesus monkeys.

In order to test the hypothesis that GABA is an inhibitory neurotransmitter restricting the release of LHRH before puberty, we examined the effects of pulsatile infusion of the GABA(A) receptor blocker, bicuculline, on the timing of puberty. Eleven female monkeys at 14-15 months of age were implanted with a stainless steel cannula into the base of the third ventricle above the median eminence. Five monkeys received bicuculline infusion every 2 h at a dose of 1 microM with a gradual increase to 100 microM in 10 microl using a portable infusion pump. The remaining 6 monkeys received similar infusions of saline. An additional 11 colony monkeys without cannula implantation were used for controls. Results indicate that bicuculline infusion advances the timing of puberty. The age of menarche (17.8+/-0.5 months) in the bicuculline infusion animals was significantly earlier than that in the saline controls (28.2+/-2.3, P < 0.001) as well as in colony controls (30.6+/-0.9, P < 0.001). The age of first ovulation (30.5+/-3.3 months) in bicuculline-treated animals was much younger (P < 0.001) than that in both controls (44.8+/-1.8 and 44.7+/-1.2, respectively). Bicuculline also accelerated the growth curve. These results suggest that the reduction of tonic GABA inhibition of LHRH neurons advances the onset of puberty.

Contextual learning requires synaptic AMPA receptor delivery in the hippocampus.

The hippocampus plays a central role in learning and memory. Although synaptic delivery of AMPA-type glutamate receptors (AMPARs) contributes to experience-dependent synaptic strengthening, its role in hippocampus-dependent learning remains elusive. By combining viral-mediated in vivo gene delivery with in vitro patch-clamp recordings, we found that the inhibitory avoidance task, a hippocampus-dependent contextual fear-learning paradigm, delivered GluR1-containing AMPARs into CA3-CA1 synapses of the dorsal hippocampus. To block the synaptic delivery of endogenous AMPARs, we expressed a fragment of the GluR1-cytoplasmic tail (the 14-aa GluR1 membrane-proximal region with two serines mutated to phospho-mimicking aspartates: MPR-DD). MPR-DD prevented learning-driven synaptic AMPAR delivery in CA1 neurons. Bilateral expression of MPR-DD in the CA1 region of the rat impaired inhibitory avoidance learning, indicating that synaptic GluR1 trafficking in the CA1 region of the hippocampus is required for encoding contextual fear memories. The fraction of CA1 neurons that underwent synaptic strengthening positively correlated with the performance in the inhibitory avoidance fear memory task. These data suggest that the robustness of a contextual memory depends on the number of hippocampal neurons that participate in the encoding of a memory trace.

The Caenorhabditis elegans Elongator Complex Regulates Neuronal α-tubulin Acetylation

Although acetylated α-tubulin is known to be a marker of stable microtubules in neurons, precise factors that regulate α-tubulin acetylation are, to date, largely unknown. Therefore, a genetic screen was employed in the nematode Caenorhabditis elegans that identified the Elongator complex as a possible regulator of α-tubulin acetylation. Detailed characterization of mutant animals revealed that the acetyltransferase activity of the Elongator is indeed required for correct acetylation of microtubules and for neuronal development. Moreover, the velocity of vesicles on microtubules was affected by mutations in Elongator. Elongator mutants also displayed defects in neurotransmitter levels. Furthermore, acetylation of α-tubulin was shown to act as a novel signal for the fine-tuning of microtubules dynamics by modulating α-tubulin turnover, which in turn affected neuronal shape. Given that mutations in the acetyltransferase subunit of the Elongator (Elp3) and in a scaffold subunit (Elp1) have previously been linked to human neurodegenerative diseases, namely Amyotrophic Lateral Sclerosis and Familial Dysautonomia respectively highlights the importance of this work and offers new insights to understand their etiology.

Sex differences in the basolateral amygdala: the extracellular levels of serotonin and dopamine, and their responses to restraint stress in rats

The sex difference in the emotional response to stress suggests a sex‐specific stress response in the amygdala. To examine the sex difference in extracellular levels of serotonin (5HT) and dopamine (DA) in the basolateral amygdala (BLA) and their responses to restraint stress, in vivo microdialysis studies were performed in male and female rats. In experiment I, dialysates were collected from the BLA at 15‐min intervals under the freely moving condition. Mean extracellular levels of 5HT or DA in the BLA were higher in male rats than in female rats. In experiment II, rats were subjected to restraint stress for 60 min to examine the stress response of 5HT or DA levels. Although restraint stress significantly increased extracellular 5HT levels in both sexes of rats, female rats showed a greater response than male rats. Moreover, restraint stress significantly increased extracellular DA levels in female rats, but not in male rats. In experiment III, rats were subjected to restraint stress for 30 min to examine behavioral responses to restraint stress. Although no sex difference was observed in the number of audible vocalizations, male rats defecated a larger number of fecal pellets than female rats. In experiment IV, rats were tested for freezing behavior to examine contextual fear responses. Conditioned male rats showed a longer freezing time than conditioned female rats. We found sex differences in the extracellular levels of 5HT and DA in the BLA and their responses to restraint stress, which may be involved in the sex‐specific emotional response to stress in rats.

Sex Differences in the Stress-Induced Release of Acetylcholine in the Hippocampus and Corticosterone from the Adrenal Cortex in Rats

To assess sex differences in stress-induced acetylcholine (ACh) release in the hippocampus and corticosterone (CS) release from the adrenal cortex, an in vivo microdialysis study was performed in intact male (n = 6) and cycling female (diestrus 1, n = 5; proestrus, n = 6) rats. Dialysates and blood samples were taken from 11.00 to 16.00 h in freely moving rats, but restraint stress was applied from 12.00 to 13.00 h. Basal ACh release in the hippocampus was low without significant differences between groups. Although ACh release promptly increased with the onset of restraint stress in both sexes, the magnitude of the increase in males was significantly greater than in female rats during diestrus and proestrus (p < 0.01). Basal serum CS concentrations were significantly greater in proestrus than in diestrus (p < 0.01) or in male rats (p < 0.01). Serum CS concentrations significantly increased at the onset of restraint stress in both sexes (p < 0.01), but the magnitude of the increase was significantly greater in female than in male rats (p < 0.01). These results indicate that sex and/or gonadal steroid environment can affect the stress-induced ACh release in the hippocampus and CS release from the adrenal cortex.

Age-related changes in diurnal acetylcholine release in the prefrontal cortex of male rats as measured by microdialysis

Extracellular levels of acetylcholine in the prefrontal cortex were measured using the micro-dialysis method in freely moving young (three to four months old) and old (23 to 24 months old) male rats over a period of 24 h to examine the effect of aging on prefrontal acetylcholine release. Prefrontal acetylcholine release during a 24 h period exhibited a diurnal variation with higher levels during the dark cycle than during the light cycle in young rats but not in old rats. In addition, prefrontal acetylcholine release was closely associated with spontaneous activity in young rats but not in old rats. The present study suggests that aging reduces diurnal changes in the prefrontal acetylcholine release and that there is a cross-correlation between the prefrontal acetylcholine release and spontaneous locomotor activity in male rats.

Gonadotropin-Releasing Hormone Exhibits Circadian Rhythm in Phase with Arginine-Vasopressin in Co-Cultures of the Female Rat Preoptic Area and Suprachiasmatic Nucleus

To determine whether the suprachiasmatic nucleus can drive a circadian release of gonadotropin‐releasing hormone (GnRH) in the preoptic area, we measured the release of GnRH, arginine‐vasopressin and vasoactive intestinal polypeptide (VIP) in cocultures of the preoptic area and the suprachiasmatic nucleus at 2‐h intervals over a period of 120 h. The release of GnRH in cocultures exhibited a significant circadian rhythm in the presence of oestrogen but not in the absence of oestrogen. The period of the GnRH circadian rhythm was the same as that of the arginine‐vasopressin circadian rhythm, and different from the VIP circadian rhythm in each coculture. Furthermore, the peak phase of the GnRH rhythm occurred at the time same as that of the arginine‐vasopressin rhythm in each coculture. However, the peak phase of the GnRH rhythm was not always the same as that of the VIP rhythm. Administration of arginine‐vasopressin significantly increased GnRH release in single preoptic area cultures in the presence of oestrogen, but VIP did not. The result suggests that, in cocultures of the suprachiasmatic nucleus and the preoptic area, arginine‐vasopressin neurones drive the circadian release of GnRH in the presence of oestrogen. We suggest that arginine‐vasopressin neurones in the suprachiasmatic nucleus mediate the clock information to GnRH neurones in vivo as well.

A cholinergic trigger drives learning-induced plasticity at hippocampal synapses

Learning induces plastic changes in synapses. However, the regulatory molecules that orchestrate learning-induced synaptic changes are largely unknown. Although it is well established that cholinergic inputs from the medial septum modulate learning and memory, evidence for the cholinergic regulation of learning-induced synaptic plasticity is lacking. Here we find that the activation of muscarinic acetylcholine (ACh) receptors (mAChRs) mediates the contextual fear learning-driven strengthening of hippocampal excitatory pyramidal synapses through the synaptic incorporation of AMPA-type glutamate receptors (AMPARs). Contextual fear learning also enhances the strength of inhibitory synapses on hippocampal pyramidal CA1 neurons, in a manner mediated by the activation of, not mAChRs, but, nicotinic AChRs (nAChRs). We observe a significant correlation between the learning-induced increases in excitatory and inhibitory synaptic strength at individual pyramidal neurons. Understanding the mechanisms underlying cholinergic regulation of learning-induced hippocampal synaptic plasticity may help the development of new therapies for cognitive disorders.

A possible role of neuropeptide Y in the control of the onset of puberty in female rhesus monkeys.

The onset of puberty is heralded by an increase in pulsatile LHRH release. Since neuropeptide Y (NPY) has been implicated as a major regulator in the control of pulsatile LHRH release in mature monkeys, we have hypothesized that maturational changes in the NPY neuronal system play an important role in puberty. To test this hypothesis, three experiments were conducted in female rhesus monkeys using a push-pull perfusion method. In the first experiment, changes in NPY release in the stalk-median eminence (S-ME) during puberty were determined in 9 prepubertal, 7 early pubertal and 8 midpubertal monkeys. NPY and LHRH levels were measured in aliquots of the same perfusate samples obtained from the S-ME. NPY release was pulsatile in all three groups. Mean NPY release and pulse frequency increased significantly from the prepubertal through the midpubertal stage. These developmental changes in NPY release were parallel to those observed for LHRH release in the same monkeys. In order to examine whether NPY infusion into the S-ME influences LHRH release during puberty, in the second experiment, NPY (10(-6) or 10(-8) M) or vehicle was infused into the S-ME for 10 min at 90-min intervals in 5 prepubertal and 9 midpubertal monkeys. In the midpubertal stage, infusion of NPY at doses of 10(-8) and 10(-6) M resulted in significant (p < 0.01) increases in LHRH release, while vehicle administration had no effect. In contrast, in prepubertal monkeys, neither NPY nor vehicle infusion altered LHRH release. In order to test whether endogenous NPY plays a role in the maintenance of pulsatile LHRH release, in the third experiment, a specific antiserum to NPY (aNPY) was infused into the S-ME of 6 prepubertal and 8 midpubertal monkeys. Infusion of aNPY (1:100, 1:1,000 dilution) significantly suppressed LHRH release in midpubertal but not prepubertal monkeys. The results are summarized as follows. (1) In prepubertal monkeys, NPY release is low, and the presence of NPY in the S-ME does not influence LHRH release. (2) At the onset of puberty, NPY release begins to increase, and NPY probably starts to stimulate LHRH release. (3) In the midpubertal period, NPY release increases further, and NPY in the S-ME is highly stimulatory to LHRH release.(ABSTRACT TRUNCATED AT 400 WORDS)

GABA release in the medial preoptic area of cyclic female rats

GABA is a potent regulator of gonadotropin-releasing hormone neurons in the hypothalamus. To determine the profile of GABA release in the medial preoptic area where the gonadotropin surge generator resides, an in vivo microdialysis study was performed in cyclic female rats. The microdialysis samples were collected and sequential blood samples (150 microl each) were also obtained, at 1-h intervals. During estrus and diestrus 1, GABA release in the medial preoptic area was relatively low. A small increase in the GABA release began in the afternoon of diestrus 1 and attained its peak in the morning of diestrus 2, but declined in the afternoon of that day. The GABA release markedly increased from late in the night of diestrus 2 through the morning of proestrus, when it attained its peak, and thereafter it declined sharply until the critical period of proestrus. A distinct preovulatory luteinizing hormone surge was observed in the afternoon of proestrus in all proestrous rats. From these results we suggest that the preovulatory elevation of the GABA release from the night through to the morning of proestrus, followed by a sharp decline, is closely associated with the onset of the preovulatory luteinizing hormone surge in cyclic female rats. The present study is the first to report the 4-day profile of GABA release in the medial preoptic area during the estrous cycle.