Neurobiology of secure infant attachment and attachment despite adversity: a mouse model.

Attachment to an abusive caregiver has wide phylogenetic representation, suggesting that animal models are useful in understanding the neural basis underlying this phenomenon and subsequent behavioral outcomes. We previously developed a rat model, in which we use classical conditioning to parallel learning processes evoked during secure attachment (odor-stroke, with stroke mimicking tactile stimulation from the caregiver) or attachment despite adversity (odor-shock, with shock mimicking maltreatment). Here we extend this model to mice. We conditioned infant mice (postnatal day (PN) 7-9 or 13-14) with presentations of peppermint odor and either stroking or shock. We used (14) C 2-deoxyglucose (2-DG) to assess olfactory bulb and amygdala metabolic changes following learning. PN7-9 mice learned to prefer an odor following either odor-stroke or shock conditioning, whereas odor-shock conditioning at PN13-14 resulted in aversion/fear learning. 2-DG data indicated enhanced bulbar activity in PN7-9 preference learning, whereas significant amygdala activity was present following aversion learning at PN13-14. Overall, the mouse results parallel behavioral and neural results in the rat model of attachment, and provide the foundation for the use of transgenic and knockout models to assess the impact of both genetic (biological vulnerabilities) and environmental factors (abusive) on attachment-related behaviors and behavioral development.

Histone acetylation in the olfactory bulb of young rats facilitates aversive olfactory learning and synaptic plasticity.

Epigenetic mechanisms play an important role in memory formation and synaptic plasticity. Specifically, histone-associated heterochromatin undergoes changes in structure during the early stages of long-term memory formation. In keeping with the classical conditioning paradigm, young rats have been shown to exhibit aversion to an odor stimulus initially presented during foot shock. We previously showed that synaptic plasticity at the dendrodendritic synapses between mitral and granule cells in the olfactory bulb (OB) underlies this aversive olfactory learning. However, the epigenetic mechanisms involved are not well characterized. Therefore, we examined whether intrabulbar infusion of trichostatin A (TSA), a histone deacetylase inhibitor, facilitates olfactory learning in young rats. TSA infusion during odor-shock training enhanced a conditioned odor aversion in a dose-dependent manner and prolonged the learned aversion. Western blot and immunohistochemical analyses showed that the level of histone H4 acetylation significantly increased until 4 h after odor-shock training in both mitral and granule cells in the OB, whereas histone H3 acetylation returned to the control level at 2 h after the training. We also obtained evidence that TSA infusion elevated acetylation of histone H4 or H3. Furthermore, in vitro electrophysiological analysis using slices of the OB revealed that application of TSA significantly enhanced the long-term potentiation induced in synaptic transmission from mitral to granule cells at dendrodendritic synapses. Taken together, these results provide evidence that histone H4 and H3 acetylation in the OB is an epigenetic mechanism associated with aversive olfactory learning in young rats.

Common properties between synaptic plasticity in the main olfactory bulb and olfactory learning in young rats.

Aversive olfactory learning was established in young rats after odor exposure paired with foot shock through a classical conditioning paradigm. Using behavioral pharmacology and Western blotting, we previously reported that plasticity in the main olfactory bulb (MOB) underlies aversive olfactory learning. Since long-term potentiation (LTP) observed in the hippocampus is believed to be a cellular substrate for aspects of memory, we attempted to induce LTP in the MOB. Using brain slices containing the MOB, we found that five tetani of the lateral olfactory tract evoked LTP that was blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist AP5. Although three tetani induced no significant changes in control slices, with noradrenaline (NA) application they produced clear LTP (NA-mediated LTP), which was not dependent on NMDA receptors. NA's facilitating effect on LTP induction was blocked by the beta-adrenoceptor antagonist timolol but not by the alpha-adrenoceptor antagonist phentolamine, and was mimicked by the beta-adrenoceptor agonist isoproterenol. The l-type calcium channel blocker nifedipine completely blocked LTP as well as NA-mediated LTP. In addition, we found that aversive olfactory learning was impaired by beta-adrenoceptor antagonist, timolol but not by alpha-adrenoceptor antagonist, phentolamine, and only odor training established olfactory learning by isoproterenol infusion. Moreover, we found that nifedipine but not AP5 prevented olfactory learning formation. These common properties provided evidence for neural correlates between NA-mediated LTP aversive olfactory learning in young rats.

Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway leading to cyclic AMP response element-binding protein phosphorylation is required for the long-term facilitation process of aversive olfactory learning in young rats.

The mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) cascade is an important contributor to synaptic plasticity that underlies learning and memory. ERK activation by the MAPK/ERK kinase (MEK) leading to cyclic-AMP response element binding protein (CREB) phosphorylation is implicated in the formation of long-term memory. We have demonstrated that CREB phosphorylation in the olfactory bulb (OB) is important for aversive olfactory learning in young rats, yet whether MAPK/ERK functions as an upstream regulator are necessary for this olfactory learning remains to be determined. Therefore, we addressed this issue using behavioral and Western blot analyses. The MEK inhibitor PD98059 was continuously infused into the OB of postnatal day 11 rat pups during a 30-min training session regarding the pairing of citral odor and foot shock. On the following day, the time spent in the part of the apparatus where the odor was present was measured as an index of odor aversion. PD98059 impaired olfactory learning in a dose-dependent manner without affecting memory retention 1 h after training. We further tested whether odor-shock training leads to MAPK/ERK activation in the OB and defines the time course of the activation. Phosphorylated ERKs (P-ERKs) 1 and 2 were significantly increased for 60 min after the training without changes in total ERKs 1 and 2. By contrast, intrabulbar infusion of PD98059 during the training significantly reduced P-ERKs 1 and 2 as well as phosphorylated CREB without any effects on the total ERKs or CREB. Taken together with the previous findings, these results indicate that the MAPK/ERK-CREB pathway is required for the long-term, but not the short-term, facilitation process of aversive olfactory learning in young rats.

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.

Non-specific olfactory aversion induced by intrabulbar infusion of the GABA(A) receptor antagonist bicuculline in young rats.

On postnatal day 12, young rats show an aversion to an odor to which they had been exposed along with presentations of foot shock on postnatal day 11. The acquisition of this aversive learning involves and requires disinhibition of the mitral/tufted cells induced by centrifugal noradrenergic activation during somatosensory stimulation. This olfactory learning is established only for the odor to which the rat has been exposed during conditioning. Infusion of the GABA(A) receptor antagonist bicuculline at a high dose (2.0 nmol/each olfactory bulb) into the olfactory bulb in the presence of an odor is capable of developing olfactory aversive responses without somatosensory stimulation in young rats. The purpose of this study is to characterize the properties of bicuculline-induced aversive responses. In contrast to the odor specificity of aversive learning produced by odor-shock conditioning, bicuculline-induced aversive responses lack odor specificity. Namely, bicuculline infusion in the presence of a citral odor results, in a dose-dependent manner, in subsequent aversive responses to strange odors (benzaldehyde and vanillin) that have never been presented. Moreover, bicuculline infusion alone is sufficient to produce dose-dependent aversive responses to strange odors (citral, benzaldehyde and geraniol). From these results we suggest that disinhibition of mitral/tufted cells from granule cells by bicuculline infusion makes young rats aversive to strange odors non-specifically, as if the rats had learned the odor aversion as a result of odor exposure paired with foot shock. Different mechanisms of disinhibition of the mitral/tufted cells may underlie both the pharmacological manipulation and noradrenergic activation by somatosensory stimulation.

Modulation of dendrodendritic interactions and mitral cell excitability in the mouse accessory olfactory bulb by vaginocervical stimulation.

When female mice are mated, they form a memory to the pheromonal signal of their male partner. The neural changes underlying this memory occur in the accessory olfactory bulb, depend upon vaginocervical stimulation at mating and involve changes at the reciprocal synapses between mitral and granule cells. However, the action of vaginocervical stimulation on the reciprocal interactions between mitral and granule cells remains to be elucidated. We have examined the effects of vaginocervical stimulation on paired-pulse depression of amygdala-evoked field potentials recorded in the external plexiform layer of the accessory olfactory bulb (AOB) and the single-unit activity of mitral cells antidromically stimulated from the amygdala in urethane-anaesthetized female mice. Artificial vaginocervical stimulation reduced paired-pulse depression (considered to be due to feedback inhibition of the mitral cell dendrites from the granule cells via reciprocal dendrodendritic synapses) recorded in the AOB external plexiform layer. As would be expected from this result, vaginocervical stimulation also enhanced the spontaneous activity of a proportion of the mitral cells tested. These results suggest that vaginocervical stimulation reduces dendrodendritic feedback inhibition to mitral cells and enhances their activity.

Reorganization of the uncrossed visual pathways as revealed by Fos-like immunoreactivity in rats with neonatal monocular enucleation.

To elucidate the neuronal characteristics of the functional expansion in the uncrossed visual pathways (UXVPs), resulting from early monocular enucleation in rats, the feasibility of stimulus-dependent induction of the immediate early gene c-fos was examined immunohistochemically. In the UXVPs of rats with monocular enucleation at birth, patterned visual stimuli induced Fos-like immunoreactive (FLI) neurons much more densely in wide areas of the superficial layer throughout the superior colliculus (SC), and in the striate and extrastriate areas of the visual cortex (VC). In the UXVPs of rats monocularly enucleated after maturity, however, only a few stimulus-dependent FLI neurons were scattered in the restricted portions of the SC and the VC.

Facilitatory effect of ritanserin is mediated by dopamine D(1) receptors on olfactory learning in young rats.

The olfactory bulb is critically involved in early olfactory learning. In this study, we examined the effect of intrabulbar infusion of ritanserin, a 5-hydroxytryptamine(2) (5-HT(2)) receptor antagonist on a one-trial aversive olfactory learning in young rats. Ritanserin, a 5-HT(2) receptor antagonist, was continuously infused into the olfactory bulb of postnatal day-11 (PND 11) rat pups during a 30-min training session of pairing citral odor and foot shock. On the following day, the time spent in the part of the apparatus where the odor was present was measured as an index of odor aversion. Consistent with a previous study on olfactory preference learning, 1 nM ritanserin, but not 10 nM, blocked the olfactory aversive learning. We further examined the ability of 10 nM ritanserin to induce olfactory learning in the absence of the unconditioned stimulus foot shock. Pups that received intrabulbar infusion of 10 nM ritanserin in the presence of citral odor developed an aversion to the odor without foot shock. Since ritanserin has been shown to have an affinity for dopamine receptors, we examined the effect of dopamine antagonists on the ritanserin-induced aversive olfactory learning. Co-infusion of the dopamine D(1) receptor antagonist (+/-)-SKF-83566 with ritanserin dose-dependently prevented induced learning. In contrast, the D(2) receptor antagonist spiperone was without effect. These results extend the previous finding on the role of bulbar 5-HT(2) receptors in early olfactory learning and suggest that high concentration of ritanserin facilitates aversive olfactory learning through D(1) receptors in the olfactory bulb.

Effect of food deprivation and leptin repletion on the plasma levels of estrogen (E2) and NADPH-d reactivity in the ventromedial and arcuate nuclei of the hypothalamus in the female rats.

The exact role of leptin in fasting has not been completely elucidated. To determine whether leptin can act in fasting to influence plasma estrogen levels and nitric oxide synthase reactivity in food regulating centers of the brain, we fasted female rats for 4 days and treated them i.p. with vehicle or 100 microg of recombinant mouse leptin as 1 ml on the 3rd and 4th day twice daily (10.00 and 17.00 h). Proestrus blood was collected at 10.00, 14.00, 18.00 and at 22.00 h, plasma obtained and assayed for estrogen (E2) and leptin levels. Verification of ovulation occurrence was by examining the oviduct for extruded ovum. The rat brains were removed and processed for nitric oxide synthase reactivity in the ventromedial hypothalamus (VMH) and arcuate nucleus (ARC) using NADPH-diaphorase histochemistry, a marker for neurons expressing NOS enzyme. Leptin effect on dependable variables such as food intake, water intake and body weight gain was also investigated. Four days fasting significantly decreased body weight, estrogen and postfast leptin levels, nitric oxide reactivity in the VMH and ARC nucleus and stopped ovulation in many (4 out of 5) rats fasted and given vehicle. Leptin treatment significantly increased plasma estrogen and postfast leptin levels, restored ovulation in many (4 out of 5) rats and increased nitric oxide reactivity in the VMH and ARC. Leptin significantly inhibited food intake, water intake and gain in body weight during recommenced feeding. These observations suggest that leptin could act in the pituitary-ovarian axis during fasting to improve reproductive function by partly stimulating estrogen secretion.

Gabaergic control of olfactory learning in young rats.

Olfactory learning in young rats correlates with neural plasticity in the olfactory bulb, and involves noradrenergic modulation of reciprocal dendrodendritic synapses between mitral cells and GABAergic granule cells. The purpose of this study was to examine, in vivo, the consequences of manipulating bulbar GABA transmission during training. In the first experiment, postnatal day 11 rat pups were trained in an olfactory associative learning task with citral odor and foot shock as the conditioned and unconditioned stimuli, respectively. The pups received continuous infusion of saline or the GABA(A) receptor agonist muscimol into the olfactory bulbs throughout a 30-min training session. The pups were then tested on postnatal day 12 for a preference for or an aversion to citral odor. Saline-infused control pups developed an aversion to citral odor. The GABA(A) receptor agonist muscimol impaired this aversive learning in a dose-dependent manner. In the second experiment, pups were exposed to the odor for 30 min while receiving continuous intrabulbar infusion of a low or high dose of the GABA(A) receptor antagonist bicuculline, without any other reinforcer. Depending on whether a low (0.2 nmol/bulb) or high (1.0 nmol/bulb) dose of bicuculline was infused, the pups showed a preference or an aversion for citral odor after infusion of low and high doses, respectively. These results indicate that disinhibition of mitral cells in the olfactory bulb is critical for olfactory learning in young rats, and suggest that the degree of disinhibition is an important determinant in acquiring either preference or aversion for the conditioned odor.

The biphasic effects of locus coeruleus noradrenergic activation on dendrodendritic inhibition in the rat olfactory bulb.

Some forms of olfactory learning require intact noradrenergic terminals in the olfactory bulb that originate from the locus coeruleus. To clarify the action of noradrenergic inputs on the dendrodendritic interaction between mitral and granule cells in the rat olfactory bulb, we analyzed field potentials in the granule cell layer of the olfactory bulb evoked by paired-pulse stimulation of the lateral olfactory tract before and after the activation of the locus coeruleus. Locus coeruleus activation by glutamate injection in the vicinity of the nucleus changed only the test response without any effect on conditioning response. Paired-pulse inhibition measured from the ratio of test response amplitude to conditioning response amplitude was significantly depressed immediately after locus coeruleus activation. Conversely, 2 min later, paired-pulse inhibition was significantly potentiated. The significant potentiation of inhibition lasted for several minutes. The depression-potentiation sequence of paired-pulse inhibition was blocked by infusion of timolol, a beta-antagonist, into the olfactory bulb, in a dose-dependent manner, but not by infusion of phentolamine, an alpha-antagonist. Infusion of isoproterenol, a beta-agonist, into the bulb mimicked the depression of paired-pulse inhibition by locus coeruleus activation. These results suggest that glutamate activation of the locus coeruleus produces a depression-potentiation sequence in granule cell-mediated feedback inhibition onto mitral cells in the olfactory bulb through beta-adrenergic receptors.

Parturition upregulates nitric oxide synthase activity in the rat anterior pituitary gland.

Recent evidence suggests that endogenous nitric oxide (NO) contributes to the modulation of hormonal secretion from the anterior pituitary gland according to the physiological state of the animal. In this study, nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and specific neurochemical assay were used to asses possible changes of NO synthase (NOS) activity in the anterior pituitary during pregnancy and parturition in rats. The anterior pituitary showed (weak) NADPH-d activity throughout pregnancy. Parturition increased the number and intensity of NADPH-d-positive cells. The NADPH-d-positive cells co-localized with immunofluorescent LH-positive cells. No variation in NADPH-d activity was apparent during the various stages of the oestrous cycle. Furthermore, NOS activity during parturition increased significantly when compared with non-pregnant and pregnant rats. Increases in both specific activity and NADPH-d activity gradually decreased within 24 h post-partum, suggesting that NO may modulate anterior pituitary function during parturition.

The effect of systemic and central nitric oxide administration on milk availability in lactating rats.

This study examined the effect of nitric oxide (NO) on milk transfer in rats. Pups nursed by mothers that received chronic systemic injections of sodium nitroprusside (SNP) weighed significantly less than pups of mothers treated with either saline or N omega-nitro-L-arginine (NNLA). Intracerebroventricular injection of SNP or L-arginine (L-arg) but not NNLA or saline, caused a significant reduction of milk transfer from mother to pups after a 12 h separation period. Systemic oxytocin (OT) injection reversed the effect of central injection of SNP. Furthermore, SNP and L-arg inhibited, whereas NNLA permitted the characteristic milk ejection burst of OT neurones without changing myoepithelial tissue response to systemic OT. These observations suggest that NO may be involved in the regulation of milk ejection bursts and milk transfer.

Nitric oxide prolongs parturition and inhibits maternal behavior in rats.

We examined the effect of nitric oxide (NO) on the process of parturition in rats. Subcutaneous injection of the NO donor sodium nitroprusside (SNP) in late pregnancy prolonged the total parturition time. The effect of N omega-nitro-L-arginine was not significantly different from that of saline. Intracerebroventricular injection of SNP in parturient rats delayed the progress of parturition. Both modes of SNP treatment also inhibited expression of maternal behavior. Central injection of oxytocin (OXT) with SNP failed to reduce parturition time significantly, but intrapartum, not postpartum, maternal behaviour was restored. These observations suggest that NO interferes with the release of OXT within the brain, hence affecting the initiation of maternal behaviour, and may also impair oxytocin secretion from the neurohypophysis.

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.

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.

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.

Influence of the electrical stimulation of the medial amygdala on adrenocortical sensitivity to adrenocorticotrophin in hypophysectomized rats.

The effects of electrical stimulation of medial amygdala on the adrenocortical sensitivity to adrenocorticotrophin (ACTH) were investigated in hypophysectomized rats. The intravenous injection of ACTH increased the rates of 14C transfer from 14C-l-acetate into corticosterone and cortisol in the adrenal slices of hypophysectomized rats. The electrical stimulation of the medial amygdala produced a further increase in the rates of 14C transfer from 14C-l-acetate into corticosterone and cortisol. From these results, it might be suggested that the medial amygdala was capable of enhancing the adrenocortical steroidogenesis in response to ACTH.