Effects of standardized Ginkgo biloba extract on the acquisition, retrieval and extinction of conditioned suppression: Evidence that short-term memory and long-term memory are differentially modulated.

Studies in our laboratory have characterized the putative neuromodulatory effects of a standardized extract of the green leaves of Ginkgo biloba (EGb), which comprises a formulation of 24% ginkgo-flavoglycosides and 6% ginkgo-terpenoid lactones, on conditioned suppression. This model comprises a suitable animal model for investigating the behavioral changes and pharmacological mechanisms that underlie fear memory and anxiety. The characterization of the effects on distinct stages of fear memory or fear extinction will help illustrate both the beneficial and harmful effects. Three hundred adult male Wistar rats were randomly assigned to 30 groups according to the treatment as follows: i-ii) control groups (CS-US and CSno-US); iii) vehicle group (12% Tween®80); and iv-vi) EGb groups (250, 500 and 1000mgkg(-1)); or experimental procedures designed to assess the effects of EGb treatment prior to the acquisition (n=20 per group) and retrieval of conditioned fear (n=10 per group) or prior to the extinction training (n=10 per group) and extinction retention test (n=10 per group). Furthermore, to better understand the effects of acute EGb treatment on fear memory, we conducted two additional analyses: the acquisition of within- and between-session extinction of fear memory (short- and long-term memory, respectively). No difference was identified between the control and treatment groups during the retention test (P>0.05), with the exception of the CSno-US group in relation to all groups (P<0.05). A between-session analysis indicated that EGb at 250mgkg(-1) facilitated the acquisition of extinction fear memory, which was verified by the suppression ration in the first trial of extinction training (SR=0.39) and the extinction retention test session (SR=0.53, P<0.05), without impairments in fear memory acquisition, which were evaluated during the retention test (SR=0.79). Moreover, EGb administered at 1000mgkg(-1) prior to conditioning did not enhance the long-term extinction memory, i.e., it did not prevent the return of extinguished fear memory in the extinction retention test, in which the spontaneous recovery of fear was demonstrated (SR=0.63, P<0.05); however, it significantly facilitated short-term memory as verified by data from the within-session extinction (1 to 8-10 trials) during the retention test (SR=0.73 to SR=0.59; P<0.05) and the extinction retention test (SR=0.63 to SR=0.41; P<0.05). Moreover, spontaneous recovery was identified in response to a higher dose of EGb when administered prior to extinction training (SR=0.75, P<0.05) and the extinction retention test (SR=0.70; P<0.05). At dose of 500mgkg(-1) EGb reduced the suppression ratio when administered prior to the retention test (SR=0.57) and extinction training (SR=0.55; P<0.05) without preventing the acquisition of fear memory, which suggests that EGb has anti-anxiety effects. Taken together, the current findings suggest that EGb differentially modulates short- and long-term memory, as well as anxiety-like behavior. The actions of EGb may provide information regarding the beneficial effects in the prevention and treatment of neurocognitive impairments and anxiety disorders. Additional analyses are necessary to facilitate an understanding of these effects; however, previous data from our group suggest that GABAergic, serotoninergic and glutamatergic receptors are potential targets of the effects of EGb on conditioned suppression.

Long-term treatment with standardized extract of Ginkgo biloba L. enhances the conditioned suppression of licking in rats by the modulation of neuronal and glial cell function in the dorsal hippocampus and central amygdala.

Our group previously demonstrated that short-term treatment with a standardized extract of Ginkgo biloba (EGb) changed fear-conditioned memory by modulating gene expression in the hippocampus, amygdaloid complex and prefrontal cortex. Although there are few controlled studies that support the long-term use of EGb for the prevention and/or treatment of memory impairment, the chronic use of Ginkgo is common. This study evaluated the effects of chronic treatment with EGb on the conditioned emotional response, assessed by the suppression of ongoing behavior and in the modulation of gene and protein expression. Male adult Wistar rats were treated over 28days and assigned to five groups (n=10) as follows: positive control (4mgkg(-1) Diazepam), negative control (12% Tween 80), EGb groups (0.5 and 1.0gkg(-1)) and the naïve group. The suppression of the licking response was calculated for each rat in six trials. Our results provide further evidence for the efficacy of EGb on memory. For the first time, we show that long-term treatment with the highest dose of EGb improves the fear memory and suggests that increased cAMP-responsive element-binding protein (CREB)-1 and glial fibrillary acidic protein (GFAP) mRNA and protein (P<0.001) in the dorsal hippocampus and amygdaloid complex and reduced growth and plasticity-associated protein 43 (GAP-43) (P<0.01) in the hippocampus are involved in this process. The fear memory/treatment-dependent changes observed in our study suggest that EGb might be effective for memory enhancement through its effect on the dorsal hippocampus and amygdaloid complex.

Operant discriminative learning and evidence of subtelencephalic plastic changes after long-term detelencephalation in pigeons.

We analyzed operant discrimination in detelencephalated pigeons and neuroanatomical substrates after long-term detelencephalation. In Experiment I, experimental pigeons with massive telencephalic ablation and control pigeons were conditioned to key peck for food. Successive discrimination was made under alternating red (variable-ratio reinforcement) and yellow (extinction) lights in one key of the chamber. These relations were interchanged during reversal discrimination. The sessions were run until steady-state rates were achieved. Experiment II analyzed the morphology of the nucleus rotundus and optic tectum in long-term detelencephalated and control birds, using a Klüver-Barrera staining and image analyzer system. Detelencephalated birds had more training sessions for response shaping and steady-state behavior (p < 0.001), higher red key peck rates during discrimination (p < 0.01), and reversal discrimination indexes around 0.50. Morphometric analysis revealed a decreased number of neurons and increased vascularity, associated with increases in the perimeter (p < 0.001) in the nucleus rotundus. In the optic tectum, increases in the perimeter (p < 0.05) associated with disorganization in the layers arrangement were seen. The data indicate that telencephalic systems might have an essential function in reversal operant discrimination learning. The structural characteristics of subtelencephalic systems after long-term detelencephalation evidence plastic changes that might be related to functional mechanisms of learning and neural plasticity in pigeons.

S100 immunoreactivity is increased in reactive astrocytes of the visual pathways following a mechanical lesion of the rat occipital cortex.

After demonstration of the paracrine action of glial neurotrophic factors, gliosis has also been considered to be related to neuronal trophism and plasticity rather than solely a repair event following brain injury. S100 is a Ca2+ binding protein, present mainly in astrocytes, that exerts paracrine trophic effects on several neuronal populations. This study analyses the presence of S100 protein by means of immunohistochemistry combined with stereology in the reactive glial cells of the rat visual pathways following a lesion of the visual cortex. Adult male Wistar rats were submitted to a unilateral aspiration of the occipital cortex or to a sham operation. One week later the rats were killed and their brain processed for immunochemistry. Single antibody immunohistochemistry was performed for the visualization of glial fibrillary acidic protein (GFAP, a marker for astrocytes), OX-42 (a marker for microglia) and S100 protein. Double immunofluorescence procedures were applied for co-localization of the S100/GFAP and S100/OX-42. An optical dissector, point interceptors and rotators were used to quantify the degree of glial activation and the changes in the S100 immunoreactivity. We observed an intense microglial and astroglial reaction in addition to an increased S100 immunoreactivity in the occipital cerebral cortex, geniculate nucleus and hippocampus ipsilateral to the lesion. In the ipsilateral superior colliculus, an intense astroglial activation was accompanied by an up-regulation of S100 immunoreactivity. Double-immunofluoresence revealed an increased S100 immunoreactivity in reactive astrocytes, but not in the reactive microglia. Evidence has therefore been obtained that after mechanical trauma, the astroglial S100 protein participates in the trophism and plasticity of the injured visual pathways.

The functional value of sound and exploratory behaviour in detelencephalated pigeons.

Exploratory behaviour in response to a sound previously paired to a shock was analyzed before and after massive telencephalic lesions. Six pigeons were submitted to sound shock associations, habituation tests, telencephalic lesion and habituation retesting. Eighteen birds divided into three sham-lesioned groups received sound shock pairing (Control 1), pre-exposure to sound stimulation (Control 2) or pre-exposure to electric shocks (Control 3) before the behavioural tests. The pre-surgery tests consisted of 25 1 s pulses of sound A (1000 Hz, 83 dB) or B (500 Hz, 85 dB), every 30 s. Retests took place 10 days after surgery using the same sequence of procedures. The behaviour exhibited immediately after each sound stimulation was recorded manually and on video during the sessions. Analysis of variance showed an effect of group {F(3, 20)= 13.37, p < 0.0001) suggesting that pre-exposure to tone-shock association affected the exploration in response to the sound presented in another context. The Experimental and Control 1 birds showed no robust decrease in responses during the pre-surgery sessions. Post-surgery exploration data for detelencephalated birds showed a lower level of responding (p < 0.05). These data suggest a retardation in the typical reduction of responses to recurrent stimulation and support other evidences of the potentiating effect of sound shock association on responding. They are also indicative of telencephalic modulation of exploratory behaviour and strengthen the arguments for the subtelencephalic storage of associative information concerning the functional value of acoustic stimulation.

Operant discrimination learning in detelencephalated pigeons (Columba livia).

Operant discrimination learning was analyzed in pigeons after massive telencephalic lesions. Twenty-one pigeons were divided into three groups: non-lesioned (N = 6), sham-lesioned (N = 5) and telencephalon lesioned (N = 10). Lesion surgeries were carried out before any experimental training. Learning procedures were run in the same sequence for all groups and under a food deprivation of 80% of the ad libitum weight. Successive discrimination was programmed by the alteration of red and yellow lights in the right key of a standard operant chamber: the red key was correlated with variable-ratio reinforcement; the yellow key was correlated with extinction. Session were run until steady-state key peck rates were obtained. The following results demonstrate discrimination learning by detelencephalated birds. Response shaping and steady-state rates required a larger number of sessions for lesioned pigeons (P < 0.05). They showed increased response rates in red (26.43 +/- 2.59) and yellow (11.17 +/- 2.86) components as compared to the non-lesioned (red: 16.51 +/- 2.0; yellow: 2.02 +/- 0.64) and sham-lesioned (red: 22.84 +/- 1.77; yellow: 4.72 +/- 1.99) groups (P < 0.05). These data show that telencephalic systems are not essential for operant discrimination learning but play a role in the modulation of discriminative behavior. Subtelencephalic systems appear to be functionally important for the organization and storage of learning.

Operant discrimination learning in detelencephalated pigeons (Columba livia)

Operant discrimination learning was analyzed in pigeons after massive telencephalic lesions. Twenty-one pigeons were divided into the three groups: non-lesioned (N=6), sham-lesioned (N=5) and telencephalon lesioned (N=10). Lesion surgeries were carried out before any experimental training. Learning procedures were run in the same sequence for all groups and under a food deprivation of 80 percent of the ad libitum weight. Successive discrimination was programmed by the alternation of red and yellow lights in the right key of a standard operant chamber: the red key was correlated with extinction. Sessions were run until steady-state key peck rates were obtained. The following results demonstrate discrimination learning required a larger number of sessions for lesioned pigeons (P<0.05). They showed increased response rates in red (26.43 +/- 2.59) and yellow (11.17 +/- 2.86) components...