Level of vigilance influences licking frequency in rats.

In rats, the basic licking rhythm is generated by the central pattern generator located in the brainstem. Nevertheless, the licking frequency can be regulated between about 7.5 and 4 Hz by changing the drinking conditions. If these conditions are kept constant, the licking frequency can be influenced only to a minor degree by factors such as deprivation level, type of solution, and phase of the session. The aim of our study was to compare the licking frequency of rats at different levels of vigilance. We investigated spontaneous licking of rats by an electrical lick sensor; parallel behavior monitoring was also performed. Animals kept in a stable environment and showing a lower level of vigilance licked at a rate of 5.96 Hz, fully vigilant rats licked significantly more rapidly at a frequency 6.57 Hz. The fastest rate of licking (6.49 Hz and 6.82 Hz, respectively) was encountered in alert rats under a mild stress caused by the presence of a second animal in the experimental box. The vigilance level is thus another factor affecting the licking rate of rats that should be taken into account in behavioral licking experiments.

CAMKII inhibition in the parabrachial nuclei elicits conditioned taste aversion in rats.

The conditioned taste aversion (CTA) paradigm was used to assess the role of Ca(2+)/calmodulin-dependent protein kinase (CAMKII) in associative learning. KN62, a specific inhibitor of CAMKII, was injected into the parabrachial nuclei (PBN) either immediately after saccharin drinking (CS) or after saccharin drinking and i.p. injection of LiCl (US). Injection of KN62 into the PBN after saccharin drinking elicited clear CTA (Exp. 1). This effect was dosage-dependent and site-specific (Exp. 2). The results are discussed in relation with an earlier report showing that CTA acquisition is disrupted by injection of Ca(2+)/phospholipid-dependent protein kinase (PKC) inhibitor chelerythrine into the PBN during CS-US interval. It is suggested that the principal serine/threonine kinases play different roles in CTA learning: whereas PKC activity is necessary for the gustatory short-term memory formation, CAMKII acts similarly to the US itself-an unexpected role of CAMKII in associative learning.

Glutamate in the parabrachial nucleus of rats during conditioned taste aversion.

Brain microdialysis combined with HPLC and spectroscopic detection was used to monitor extracellular glutamate in the parabrachial nucleus (PBN) of rats during acquisition of a conditioned taste aversion (CTA). Microdialysis fractions taken every 20 min were used to assess the effects of presentation of the conditioned stimulus alone (CS, consumption of 0.1% saccharin), the unconditioned stimulus alone (US, intraperitoneal injection of 0.15 M LiCl, 2% b.w. induced malaise after water drinking) as well as that of CS-US pairing. After 15 min of saccharin drinking, the glutamate concentration in the eluate (20 microl/20 min) reached 80% above the baseline but returned to the basal value in the next fraction. LiCl alone (applied 1 h after 15 min drinking of water) increased glutamate only following some delay, i.e. in the second and third post-lithium fraction by 90 and 67%, respectively. However, when LiCl was injected 1 h after the onset of saccharin intake, the glutamate concentration rose significantly (by 95%) already in the first post-LiCl fraction and by 120% in the second one. It appears, therefore, that the 'saccharin trace' facilitates the effect of lithium on extracellular concentration of glutamate in PBN during acquisition of CTA.

Chelerythrine, a specific PKC inhibitor, blocks acquisition but not consolidation and retrieval of conditioned taste aversion in rat.

Association of the short-term memory of the gustatory conditioned stimulus (CS) with visceral malaise (unconditioned stimulus, US) in conditioned taste aversion (CTA) paradigm takes place in the parabrachial nuclei (PBN) of brainstem. In order to ascertain the role of protein-kinase C (PKC) during different phases of CTA acquisition and retrieval, four experimental series were carried out. In Experiment 1, 1 microl of 10 mM of PKC inhibitor chelerythrine prevented CTA acquisition when applied into PBN in the CS-US interval. In Experiment 2, the necessity of PKC activity in different phases of CTA acquisition was tested by prolonging the time interval between PBN administration of chelerythrine and i.p. LiCl. CTA acquisition was prevented when chelerythrine-induced blockade of PKC coincided with GSTM persistence but not with CTA consolidation. In Experiment 3, the interval between saccharin drinking and LiCl injection was prolonged to 120 min. Again, chelerythrine blockade of PKC activity prevented CTA formation when it interfered with GSTM persistence. In Experiment 4, the possibility that PKC activity is necessary also for CTA retrieval was tested by chelerythrine application into PBN 5 min before retrieval testing. In this case, the chelerythrine-induced PKC blockade did not impair CTA retrieval. It is concluded that PKC is important for GSTM formation and persistence but not for CTA consolidation or retrieval.

Tetrodotoxin inactivation of the gustatory cortex disrupts the effect of the N-methyl-D-aspartate antagonist ketamine on latent inhibition of conditioned taste aversion in rats.

The effect of the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist ketamine on latent inhibition of taste aversion learning was studied in rats. Systemic injections of ketamine (50 mg/kg) applied after each of three preexposures to sodium saccharin (0.1%) disrupted the latent inhibition effect. The blockade was not due to aversive properties of ketamine, because three saccharin-ketamine pairings did not produce saccharin aversion. Moreover, the ketamine-induced blockade of latent inhibition was disrupted by tetrodotoxin injections (10 ng/microl)-induced reversible inactivation of gustatory cortex, applied after each preexposure. A specific gustatory cortex mediation of the ketamine effect is discussed.

Interaction with demonstrator rats changes observer rats' affective responses to flavors.

The authors examined whether exposing naive rats (observers) to recently fed conspecific demonstrator rats changed the observers' later affective responses to foods their demonstrators ate. In Experiment 1, observers learned an aversion to a flavored fluid, then interacted with demonstrators that had drunk that fluid. These observers, but not those interacting with demonstrators that had drunk water, increased their intake of the averted fluid and exhibited fewer negative responses when the averted fluid was infused into their mouths. Rats in Experiment 2 entered the arm of a T maze known to lead to banana-flavored pellets more frequently after interacting with demonstrators fed banana-flavored pellets than after interacting with demonstrators fed chow-flavored pellets. Results of both experiments indicated that interaction with demonstrator rats changed observer rats' affective responses to flavors.

Ipsilateral connections between the gustatory cortex, amygdala and parabrachial nucleus are necessary for acquisition and retrieval of conditioned taste aversion in rats.

Acquisition and retrieval of conditioned taste aversion (CTA) is implemented by the interaction of several brain structures. In order to clarify the role of 3 important gustatory and visceral relays, gustatory neocortex (GC), amygdala (AM) and parabrachial nucleus (PB), two experimental series were carried out. In Expt. 1 reversible unilateral lesions of the PB and AM or of AM and GC were induced by 10 ng of tetrodotoxin (TTX) either in the same hemisphere or in the opposite hemispheres during CTA acquisition. It was found that contralateral TTX injections into the above structures caused a complete blockade of CTA acquisition which was not affected by ipsilateral TTX administration. In Expt. 2, retrieval of CTA acquired during unilateral blockade of the PB or AM in one hemisphere has been examined during unilateral TTX blockade of GC alone, AM alone or during combined GC and AM inactivation either in the same or in the contralateral hemispheres. A clear CTA retrieval impairment was found only when structures in the opposite hemispheres were blocked. These results indicate a complete lateralization of CTA acquisition and retrieval processes and suggest that the long-term CTA engram can be formed in each hemisphere separately without participation of the contralateral hemisphere.

Intracerebral injection of polymyxin B blocks the acquisition of conditioned taste aversion in rats.

The contribution of protein kinase C (PKC) to the acquisition of conditioned taste aversion (CTA) was tested by injection of three PKC inhibitors--polymyxin B, H7 and staurosporine--into the parabrachial nucleus (PBN). From the tested drugs only polymyxin B (20 mM) prevented CTA acquisition. Application of H7 (10 mM) and staurosporine (100 and 500 microM) into the PBN did not impair CTA learning. The blocking effect of polymyxin B is dose dependent (5 and 10 mM concentration did not disrupt CTA formation) and site specific (application of polymyxin B into the visual cortex did not elicit CTA blockade). The ability of polymyxin B to disrupt CTA learning is not due to irreversible damage of PBN. These results suggest that polymyxin B blocks acquisition of CTA in some nonspecific way not necessarily involving inhibition of PKC. This conclusion is supported by failure of two other more specific PKC inhibitors to affect CTA learning.

Universality of parabrachial mediation of conditioned taste aversion.

Disruption of conditioned taste aversion (CTA) by tetrodotoxin (TTX) blockade of parabrachial nuclei elicited after ingestion of the gustatory CS and before administration of the visceral US is revealed by unchanged CS preference in retrieval test performed two days after acquisition. The universality of the above amnesia was examined in rats. In Experiment 1, parabrachial TTX disrupted CTAs when sodium saccharin (0.1%), quinine hydrochloride (0.0072%), sodium chloride (0.9%) or a garlic extract were used as the CS and i.p. LiCl (0.15 M, 2% body weight) as the US. In Experiment 2, parabrachial TTX disrupted CTAs when 0.9% NaCl was used as the CS and i.p. injection of LiCl (0.15 M, 2% body weight), D-amphetamine (3 mg/kg) or carbachol (0.15 mg/kg) served as the US. Experiment 3 showed that post-drinking parabrachial TTX blocked also CTA induced by LiCl drinking, i.e. under conditions of CS-US identity. Experiments 4 and 5 demonstrated that the amnesia is not confounded by TTX-induced reduction of fluid intake because the CTA disruption was well expressed even when tested after recovery of normal fluid intake 3 days after TTX injection and in animals receiving 12 days of preacquisition adaptation to limited fluid access. It is concluded that parabrachial nuclei represent an important point of convergence for various classes of CTA eliciting stimuli.