Fos protein expression induced by intracerebroventricular injection of vasopressin in unconditioned and conditioned mice.

Arginine8-vasopressin (AVP) has been shown to improve memory consolidation in various mnemonic tasks. Our previous studies have pointed out the involvement of the hippocampus in memory consolidation and retrieval processes during discriminative learning by mice. The present study attempts to determine what other brain areas besides the hippocampus might be involved in the enhancing effect of intracerebroventricularly (i.c.v.) injected AVP on memory consolidation in a visual discrimination task using a polyclonal antibody that acts against Fos and Fos-like proteins. For behavioral testing, AVP was i.c.v. injected at the behaviorally active dose of 2 ng after the last learning session and improvement in consolidation processes was assessed in a retention session. Changes in Fos and Fos-like protein expression were determined in non-conditioned and conditioned mice. In non-conditioned mice, AVP i. c.v. injected at a dose of 2 ng evoked a time-dependent increase in Fos and Fos-like protein expression in the dentate gyrus (DG), CA1 and CA3 hippocampal fields, lateral septum (LS), bed nucleus of the stria terminalis, and basolateral and central amygdaloid nuclei, with a peak 120 min after the injection in most of the these brain areas. In contrast, in conditioned mice, an increase in the level of Fos expression, assessed 120 min after the end of learning and the injection of AVP, was detected only in the DG, ventral CA3 hippocampal field, and LS. Thus, the pattern observed after post-training injection of AVP was not the same as that evoked by AVP alone, since among the limbic structures activated following AVP alone, only the DG, the CA3 hippocampal field, and the LS seem to be involved in the enhancing effect of AVP on memory consolidation in discriminative learning.

The behavioral effect of vasopressin in the ventral hippocampus is antagonized by an oxytocin receptor antagonist.

[Arg8]vasopressin improved long-term retrieval processes and relearning in a go-no go visual discrimination task when bilaterally microinjected at a dose of 25 pg/animal into the ventral hippocampus of mice, 10 min prior to the retention session. We had shown that this enhancing effect is antagonized by pretreatment with equal or lower doses (25 pg or 1 ng) of the vasopressin V1 receptor antagonist, (d(CH2)5Tyr(Me)-vasopressin). The present study was an attempt to determine whether the vasopressin V2 receptor antagonist or oxytocin receptor antagonist is as effective as the vasopressin V1 receptor antagonist to block the behavioral effect of vasopressin in the ventral hippocampus. We tested the effect of 25 pg of [d(CH2)5-D-Ile2,Ile4,Arg8]vasopressin, a vasopressin V2 receptor antagonist, and [d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH9(2)]ornithine vasotocin, an oxytocin receptor antagonist, under the same experimental conditions as those used to test the effect of the vasopressin V1 receptor antagonist. The results showed that the vasopressin V2 receptor antagonist microinjected into the ventral hippocampus did not alter the enhancing effect of vasopressin on retrieval and relearning. In contrast, the oxytocin receptor antagonist blocked the vasopressin-enhancing effect on retention processes. We can conclude from the data that both vasopressin V1 receptors and oxytocin receptors seem to be involved in the enhancing effect of vasopressin on memory retention. In contrast, the vasopressin V2 receptors do not seem to be involved in the effect of the peptide.

Effects of arginine8-vasopressin administered at different times in the learning of an appetitive visual discriminative task in mice.

A visual discrimination task was used to investigate the effect of the intra-hippocampal injection of arginine8-vasopressin (AVP) in male Balb/c mice at different stages of the learning processes. The peptide was bilaterally microinjected at a dose of 25 pg per animal, i.e. 833 pg/kg, into the ventral hippocampus, in a volume of 0.3 microliter 10 min before either the first or the second learning session, or immediately after the first or second learning session. Following pre-session administration of AVP, no effect of the peptide was observed on the session prior to which it was administered. On the other hand, 48 h after the pre-first session treatment, it seems that AVP animals had trouble learning the task. Following post-session injection of AVP, no effect was observed when the treatment was given after the first learning session and a tendency to improve performance was noted when the treatment was given after the second learning session. Thus, whatever time AVP was injected during learning, little or no effect was observed. These results and previous work on the same behavioral task showing a clear enhancing effect of the peptide on retrieval processes, suggest that prior experience or mnemonic context before AVP treatment is as important a factor in understanding the effects of AVP on memory processes as the administration route or the doses used.

Neonatal gamma-ray irradiation impairs learning and memory of an olfactory associative task in adult rats.

Adult neonatally gamma-irradiated rats were compared with control animals in a non-spatial olfactory associative task using two different procedures. Irradiation induced a clear reduction in the total mean area of the olfactory bulbs and hippocampus but not of the orbital prefrontal cortex, diagonal band and cell layers of the entorhinal and piriform cortex. The gamma-irradiation affected the granule cells of the olfactory bulbs and differentially altered the cell layers of the subfields of the ammonic fields and the dorsal and ventral blades of the dentate gyrus. In the CA1 ammonic field, dorsal and ventral blades of the dentate gyrus, the cellular loss was significant in comparison with control adult rats. The behavioural data indicated that irradiated rats were deeply disturbed in learning the odour-reward association, and substantially impaired in a reversal experiment, but not in the discrimination of the odours per se. The cellular loss in the olfactory bulbs, in the CA1 and in the ventral blade of the gyrus dentatus was positively correlated with the deficit in behavioural performance. The data support the findings that the hippocampal system participates in the odour-reward associations and facilitates the long-term storage of associations after learning is achieved in this olfactory associative task.

Distribution of cholecystokinin immunoreactivity in the BALB/c mouse forebrain: an immunocytochemical study.

The present study describes cholecystokinin (CCK) immunoreactivity (CCK-IR) distribution in the brains of control and colchicine-treated mice. In the brains of control mice, the CCK-IR strongly revealed numerous axons and terminals. Perikarya exhibiting a faint to moderate immunoreactivity were also observed in areas such as cortices, hippocampus, amygdala, septum, and thalamus. The colchicine treatment did not seem to notably affect the brain CCK-IR innervation, but resulted in profound changes of the perikaryal staining. Indeed, the regions, which contained numerous moderately stained perikarya in the control animals, exhibited after colchicine treatment immunoreactive perikarya intensely stained but only in moderate number. This feature obviously appeared in the cortex in which, in addition to strongly stained perikarya, colchicine induced the appearance of numerous CCK-IR hillocks. In the lateral amygdala and thalamus of colchicine-treated animals, the somatic immunoreactivity was considerably decreased. The regions, such as paraventricular hypothalamic nucleus and bed nucleus of the stria terminalis, which in the control animals did not exhibit any stained perikaryon, showed a high number of strongly stained cell bodies after colchicine treatment. This study, mapping the mouse forebrain CCK-IR, demonstrated a wide distribution of this peptide. Moreover, CCK-IR is spontaneously visible in neurons of untreated mouse in some brain areas previously shown in the rat to exhibit CCK mRNA, but no clear perikaryal CCK-IR even after colchicine treatment.

Inhibition of the vasopressin-enhancing effect on memory retrieval and relearning by a vasopressin V1 receptor antagonist in mice.

We have previously shown that [Arg8]vasopressin bilaterally administered into the ventral hippocampus of mice at a dose of 0.025 ng/animal 10 min prior to the retention session, improved long-term retrieval processes and relearning of a Go-No-Go visual discrimination task. The purpose of the present study was to determine whether the vasopressin V1 receptor antagonist, -beta-mercapto-beta,beta-cyclopentamethylenepropionyl1, O-Me-Tyr2,Arg8]vasopressin, d(CH2)5Tyr(Me)vasopressin), is able to block the behavioral effect of arginine-vasopressin in the ventral hippocampus. We first tested the effect of three doses of d(CH2)5Tyr(Me)vasopressin (0.025, 1, and 6.3 ng/animal) in the same experimental conditions as used for arginine-vasopressin. The results showed a dose-dependent deleterious effect of the vasopressin V1 receptor antagonist on retrieval and relearning, suggesting the involvement of endogenous arginine-vasopressin in the ventral hippocampus for these memory processes. Second, we tested the ability of d(CH2)5Tyr(Me)vasopressin to block the enhancing effect of experimentally administered arginine-vasopressin. The antagonist was injected at a dose of 0.025 ng, which had no intrinsic effect on behavior, or at a dose of 1 ng, which had a weak deleterious effect on behavior, followed by administration of 0.025 ng of arginine-vasopressin. The results showed that even at the weakest dose (0.025 ng), d(CH2)5Tyr(Me)vasopressin blocked the enhancing effect of arginine-vasopressin on retrieval and relearning. Thus, as for other behaviors and structures, the antagonist microinjected into the ventral hippocampus prevents the enhancing effect of arginine-vasopressin on long-term retrieval and relearning. However, the exclusive involvement of the vasopressin V1 receptors remain to demonstrate vis-a-vis oxytocin receptors.

Effects of anti-CCK-8 antiserum on acquisition and retrieval by mice in an appetitive task.

Endogenous cholecystokinin (CCK) was blocked in the posterior cingulate cortex of BALB/c mice using a local injection of anti-CCK-8 antiserum, and memory effects were tested using Go-No Go visual discrimination conditioning. Injection of 0.4 microliter of anti-CCK-8 antiserum diluted to 1:10, 10-15 min before each session, produced substantial learning impairment on the discrimination task. But when injections were stopped, animals began to learn the task normally, showing that the anti-CCK antiserum effect was reversible. When the antiserum was administered at the same dose before a single test session 14 days after the end of the initial training, the retrieval process was also disturbed. These results show that cingulate cholecystokinin is essential for memory processes and suggest that cholecystokinin octapeptide may be a neuromodulator of the thalamo-cingulate pathway of Papez's circuit, which is involved in memory processes.

Effect of changes in the intrahippocampal vasopressin on memory retrieval and relearning.

Previous results have indicated the involvement of the hippocampus in the behavioral effect of vasopressin, with a better effect when the peptide was injected in the ventral part rather than in the dorsal part of this structure. The purpose of the present study was to determine, in mice, whether the injection of vasopressin or vasopressin antisera into the ventral hippocampus has an effect on retrieval and relearning of a Go-No Go visual discrimination task and, if so, to what extent this involvement of the vasopressin system depends on the integrity of the medial amygdaloid nucleus, the main source of vasopressin innervation in the ventral hippocampus in rats. In the first experiment, we showed that pretest microinjection of Arg8-vasopressin (25 pg per animal) in the ventral hippocampus alleviated forgetting observed after a prolonged interval of 24 days between the acquisition of information and its retrieval. This enhancing effect was characterized by better retrieval and relearning in vasopressin-treated mice than those in control mice. Conversely, an immunoneutralization of endogenous vasopressin in the ventral hippocampus by the microinjection of vasopressin antisera (1/10 dilution) resulted in the drastic impairment of retrieval and relearning. Since the lack of an observable change in a locomotor activity test might explain these results, we postulated that the vasopressin system in the ventral hippocampus is involved in retrieval processes. Moreover, the effects of these treatments in a nonassociative context suggest that the effect of vasopressin could be dependent on the contextual paradigm used. In the second experiment, we localized vasopressin immunoreactive fibers in the CA1-CA2 ventral hippocampal fields and CA4-gyrus dentatus region, and vasopressin perikarya in the medial amygdaloid nucleus. Then, the projection of vasopressin cells from the medial amygdaloid nucleus to the ventral hippocampus was evaluated by studying changes in vasopressin immunoreactive fiber density in the ventral hippocampus after a lesion of the medial amygdaloid nucleus. The results showed the almost complete disappearance of vasopressin fibers in the CA1-CA2 hippocampal fields after the medial amygdaloid lesion. In contrast, vasopressin fibers in the CA4 and gyrus dentatus region remain unchanged. On the basis of our immunohistochemical results, our third experiment tested the repercussions of the change in vasopressin innervation in the ventral hippocampus, due to the medial amygdaloid lesion, on the effects of exogenously administered vasopressin on both retrieval and relearning processes. The medial amygdaloid lesion induced a deleterious effect on retrieval without really affecting the ability to relearn. No observable change in locomotor activity could explain this impairment.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of bilateral lesions of the cerebellar interpositus nucleus on the conditioned forelimb flexion reflex in mice.

Three groups of mice, unoperated controls, sham and lesioned, were submitted to an associative conditioning of forelimb flexion reflex (FFR). Light and tone constituted the conditioned stimulus (CS) paired with a forelimb electric shock, the unconditioned stimulus (UCS). The first two groups were able to acquire an appropriate conditioned response. In the third group, each animal received a bilateral lesion of the cerebellar interpositus nucleus (IN). The subjects of this group were unable to acquire the conditioning. When bilateral lesions of the IN were done after the acquisition, no effect of the lesions could be detected during retention test sessions 10 days after surgery, by comparison with sham controls. It is therefore concluded that the cerebellar interpositus nucleus is an essential part of the circuit for the acquisition of associative conditioning of the forelimb flexion response in mice, but not for the retention of this task. Moreover, no direct sensorimotor effect of the lesion on performance itself could be evoked.

Microinjection of anti-vasopressin serum into hippocampus in mice: effects on appetitively reinforced task after intraventricular administration of Arg-vasopressin.

Antiserum to [Arg8]vasopressin (anti-AVP) was bilaterally administered into dorsal hippocampus at 1:50 or 1:10 dilution 20 min before the 24-day retention session of a visual discrimination task. This treatment by itself did not affect the retention performance by comparison with the respective control group, whatever the dilution of anti-AVP, suggesting that hippocampal endogenous AVP is not involved in our behavioral paradigm. On the other hand, intracerebroventricular (i.c.v.) administration of AVP 10 min before the retention session improved retention performance of the visual discrimination task. When anti-AVP was injected at the 1:10 dilution into the dorsal hippocampus 10 min before the i.c.v. administration of AVP, the retention performance was not improved. These data suggest the involvement of the hippocampus in the behavioral expression of AVP following an i.c.v. treatment.

Hippocampal lesions block behavioral effects of central but not of peripheral pre-test injection of arginine vasopressin in an appetitive learning task.

The effects of peripheral and central administration of arginine-vasopressin (AVP) were examined in intact or hippocampal-lesioned mice, when administered just before the retention session of an appetitive visual discrimination task. All the subjects underwent a partial learning of this task and were tested 24 days later when a partial forgetting occurred in controls. In Expt. 1, intact mice received AVP just before the test session either subcutaneously (s.c., 1 microgram) or intracerebroventricularly (i.c.v., 1 ng). Both routes of administration resulted in an enhancement of the retention performance. In Expt. 2, bilateral lesions of the dorsal hippocampus were carried out 2 days after the end of initial learning. The lesions did not modify retrieval performance on the 24th day. In additional groups, AVP was injected in pre-test situation in lesioned animals. An s.c. AVP injection induced the same improvement of retrieval as observed in intact mice. On the contrary, i.c.v. administration of AVP accentuated forgetting in lesioned animals. In other words, hippocampal lesions reversed the effects of i.c.v. pre-test administration of AVP. The results are analyzed in terms of different mechanisms for both routes of administration of AVP.

Age-related changes in the C57BL/6J mouse cochlea. II. Ultrastructural findings.

The C57BL/6J mouse organ of Corti was studied using transmission electron microscopy. The basal coil of the cochlea was examined in mice 1-50 days of age. At birth the cochlea was very immature but both types of hair cells were innervated. Inner hair cells (IHC) were connected to afferent and efferent processes, and efferent endings synapsed on IHC afferents. Outer hair cells (OHC) were innervated exclusively by afferents, which made well-defined synapses with the cell. Maturation at IHC took place rapidly and was essentially over by about 12 days of age. Changes were largely restricted to an increase in presynaptic specializations opposite afferents and a decrease in postsynaptic specializations (cisterns) opposite efferents. Ontogeny at OHC took place over a longer, 2.5 week, period. Afferent synapses lost their presynaptic specializations (synaptic bodies), and then efferent fibers arrived below OHC. The efferents subsequently made temporary axo-dendritic synapses with the afferents before replacing most of them at OHC. The first synapses between efferent endings and OHC were seen at 9 days of age, but it was not until about 20 days of age that mature synapses were seen. Some evidence of hair cell degeneration was seen in 30- and 50-day-old mice. The results are discussed in terms of sensory hair cell differentiation, the disappearance of OHC synaptic bodies, and age-related changes in auditory system function.

Significance of presynaptic formations in early stages of cochlear synaptogenesis.

Very early stages of cochlear synaptogenesis are described in inner (IHCs) and outer hair cells (OHCs) of cat foetuses. Ten days before birth (DBB) well-formed synaptic contacts were found between afferent dendrites and both IHCs and OHCs. At the IHC level a preliminary stage before functioning has been proposed. But at the OHC level where the adult cell membrane becomes mainly postsynaptic, we could only formulate hypotheses based on phylogenetic considerations (afferents precede efferents), or on embryology (presynaptic specializations play a role in the arrival and growth of fibers around the hair cell).

Different patterns of cochlear innervation during the development of the kitten.

The postnatal development of neuro-epithelial junctions inside the kitten cochlea has been investigated by electron microscopy, and correlated with previous electrophysiological results. Two main stages of development are described. During the first postnatal days, outer hair cells look very immature with only a few afferent endings adjoining them. The inner hair cells, on the other hand, are surrounded by numerous endings with mature afferent and efferent synapses. Thus, when the efferent olivo-cochlear system begins to function during the first postnatal day s, it is able to modify only inner hair cell responses. The second postnatal week is characterized by maturation of the large efferent endings below the outer hair cells. At the same time, direct efferent connections become sparce at the level of inner hair cells. The maturation of hearing, at the receptor level, seems to proceed in two steps, one related to inner hair cells and corresponding to a gross and primitive hearing, the other related to outer hair cells and corresponding to more precise and discriminative hearing abilities.