Overexpression of hippocampal Ca2+/calmodulin-dependent protein kinase II improves spatial memory.

Hippocampal alpha-calcium/calmodulin-dependent protein kinase II (alphaCaMKII) has been implicated in neuronal plasticity and spatial learning. In the present experiment, an adeno-associated virus (AAV) vector was designed to express alphaCaMKII driven by the U6 promotor. Microinfusion of this vector into the rat hippocampus increased alphaCaMKII immunoreactivity by approximately 73% (Western analysis) and improved performance in a water maze task. Locomotor activity and exploratory behavior in an open field task were not altered by the overexpression of alphaCaMKII. These data support a role for alphaCaMKII in spatial or explicit memory storage. The advantages of viral vectors for manipulating target proteins expression compared with genetically modified mouse models are discussed.

In vivo inhibition of hippocampal Ca2+/calmodulin-dependent protein kinase II by RNA interference.

Hippocampal alpha-Ca2+/calmodulin-dependent protein kinase II (alpha-CaMKII) has been implicated in spatial learning, neuronal plasticity, epilepsy, and cerebral ischemia. In the present study, an adeno-associated virus (AAV) vector was designed to express green fluorescent protein (GFP) from the CBA promoter and a small hairpin RNA targeting alpha-CaMKII (AAV-shCAM) driven from the U6 promoter. The AAV-shCAM or control vector was microinfused into the rat hippocampus and behavioral testing conducted 19-26 days following surgery. Expression of the marker gene and alpha-CaMKII was evaluated 31 days following AAV infusion. GFP expression was localized to the hippocampus and extended +/-2 mm rostral and caudal from the injection site. Hippocampal alpha-CaMKII was significantly reduced following AAV-shCAM treatment as demonstrated using immunohistochemical and Western analysis. This suppression of alpha-CaMKII was associated with changes in exploratory behavior (open field task) and impaired place learning (water maze task). These results demonstrate the efficacy of a viral-based delivered shRNA to produce gene suppression in a specific circuit of the brain.

In vivo glutamate neurotoxicity is associated with reductions in calcium/calmodulin-dependent protein kinase II immunoreactivity.

Calcium/calmodulin-dependent protein kinase II (CaM kinase) activity is inhibited in cultured hippocampal cells following direct application of glutamate. The goal of the present study was to determine if hippocampal regions that undergo delayed cell death following glutamate microinfusion would exhibit changes in CaM kinase immunoreactivity. Gerbils received bilateral intra-hippocampal infusions of L-glutamate (34 microg/microl), or control treatments of D-glutamate or saline. Animals were sacrificed at 12 or 24 hr to assess cell loss and determine changes in CaM kinase-like immunoreactivity. Hippocampi of gerbils euthanized 12 hr following L-glutamate, or 24 hr following D-glutamate, did not exhibit cell death in the hippocampal CA1 region. Animals injected with L-glutamate and sacrificed 24 hr after infusion had extensive cell damage that was restricted to the hippocampal CA1 region. CaM kinase-like immunoreactivity was absent in the hippocampal CA1 region of all L-glutamate treated animals sacrificed at 12 hr. In these same sections, CaM kinase immunoreactivity was evident in the subiculum, CA2 and CA3 regions. Reduction in CaM kinase immunoreactivity following L-glutamate were also observed using Western analysis. The results confirm and extend the findings of earlier cell culture studies by demonstrating a reduction in CaM kinase immunoreactivity that occurred prior to cell death.

Importance of preoperative training and maze difficulty in task performance following hippocampal damage in the gerbil.

A series of experiments was conducted to evaluate the effects of preoperative training on three variations of a spatial working memory task following ischemic damage to the hippocampus. A discrete trial, pair-run procedure in a standard T-maze apparatus was used. Ischemic and sham gerbils were tested on a Win/Shift, Win/Shift Delay (10 s), or Win/Stay task with or without preoperative training. Gerbils tested on the Win/Shift or Win/Shift Delay tasks did not exhibit improved performance as a result of pretraining. Ischemic gerbils in Win/Shift tasks exhibited working memory impairment with or without preoperative training. In contrast, pretraining was found to significantly improve the performance of gerbils tested in the Win/Stay task. Choice accuracy of pretrained ischemic gerbils was not significantly different from controls. These data suggest that preoperative training can influence performance, however the difficulty of the task and/or the amount of pretraining are important variables. The tendency for rodents to alternate makes the Win/Shift task ideal when extensive training or testing is not possible.

Transient cerebral ischemia decreases calcium/calmodulin-dependent protein kinase II immunoreactivity, but not mRNA levels in the gerbil hippocampus.

During transient cerebral ischemia, intracellular calcium increases initiating a cascade of events which leads to the delayed death of neurons located in the hippocampus. Coupled to this calcium disturbance is the rapid decrease of calcium/calmodulin kinase II (CaM kinase) activity, a protein kinase critical to neuronal functioning. The present study correlated the increased locomotor activity following ischemic insult with alterations in CaM kinase mRNA levels and immunocytochemical labeling of alpha and beta CaM kinase subunits in the hippocampus. The protective effect of hypothermia was also compared with CaM kinase mRNA levels and immunoreactivity. Levels of CaM kinase message for either alpha or beta subunits was not altered in ischemic gerbils compared to sham or hypothermic ischemic conditions. Immunoreactivity for both the alpha and beta subunits was markedly reduced in the vulnerable CA1 region of ischemic animals compared to sham controls. Gerbils that underwent the ischemic insult while hypothermic showed no decrement in staining. CaM kinase-like immunoreactivity in the ischemia-resistant CA3 sector was not altered following ischemia. These data suggest that the loss of hippocampal CaM kinase immunoreactivity observed at 24 h following ischemia is not associated with a reduction in CaM kinase mRNA levels and support the notion that the rapid decline in CaM kinase activity following ischemic insult is a result of a posttranslational modification and/or translocation of the enzyme.

Neuropeptide Y attenuates satiety: evidence from a detailed analysis of patterns ingestion.

Centrally injected neuropeptide Y (NPY) is a potent stimulant of ingestive behavior capable of augmenting both food and fluid intake in fully satiated animals. To gain further insight into NPY's mechanism of action, we recorded patterns of licking behavior in rats drinking sweetened condensed milk solutions immediately after lateral ventricular injection of NPY (10 micrograms) or vehicle. In a separate study, we examined licking patterns after 23 h food deprivation (FD) that produced approximately the same total intake as NPY. Consistent with previous reports, we found NPY stimulated intake by increasing total ingestion time and total volume consumed during a 1-h test. Although NPY increased the number of bouts of licking and shortened pauses between bouts, it also decreased mean bout size, bout duration and within-bout lick rate (local rate). It had no significant effect on start latency or lick efficiency (licks/ml). Further analyses revealed that NPY attenuated satiety (reduced slope of lick-rate functions with session time) but had no significant effect on the beginning lick rate, a measure related to orosensory excitation. In contrast to NPY, FD increased both the beginning lick rate and individual bout size without changing either the mean number of bouts or the pause between bouts. In general, NPY stimulated an intermittent pattern of licking and delayed satiation whereas FD increased the initial rate of licking and the size of individual bouts without changing the basic licking pattern. The increase in initial lick rate suggests that FD, unlike NPY, enhances orosensory stimulation. These data compliment previous results showing that NPY increases the motivation to eat.(ABSTRACT TRUNCATED AT 250 WORDS)

Locomotor activity in the ischemic gerbil.

Previous studies have shown that within 24 h after ischemic insult, gerbils exhibit an increase in locomotor activity. Because activity gradually diminishes to normal levels with repeated testing, it has been argued that this behavior represents a reversible or transient effect of ischemia. The present study challenged this notion by testing ischemic gerbils at a time when increased activity is not observed with repeated testing. Ischemic (5-min bilateral carotid occlusion) and sham gerbils were tested for 14 consecutive days after reperfusion in an open-field apparatus (n = 6/condition). As previously reported, ischemic gerbils exhibited a significant increase in activity (days 1 and 2) which returned to control levels with repeated testing (days 13 and 14). A second group of ischemic and sham gerbils (n = 6/condition) were tested only on days 13 and 14 after reperfusion. In contrast to those tested repeatedly, these ischemic gerbils displayed increased locomotor activity as compared with sham controls. In addition, gerbils in the repeated testing conditions were evaluated in a semi-novel testing environment on days 15 and 16 after surgery. The locomotor activity of ischemic gerbils significantly increased in response to the semi-novel environment. These results suggest that the effects of ischemia on locomotor activity are not limited to a brief period after occlusion and may represent a permanent deficit. In addition, as previously suggested, this behavior may represent a deficit in habituation or spatial mapping rather than motor hyperactivity.

Effects of bombesin on temporal patterns of ingestion in the rat.

Bombesin, an analog to gastrin releasing peptide, has previously been shown to inhibit food intake in the rat. In order to further characterize the effects of bombesin on ingestive behavior, the present study examined licking patterns of rats drinking sweetened condensed milk following bombesin (4 micrograms/kg, IP) or vehicle injection under two levels of food deprivation (0 and 24 h). Both bombesin treatment and satiety (reducing food deprivation from 24 to 0 h) significantly decreased total milk consumption during a 1-h test. Analysis of licking patterns suggested that bombesin and satiety operate by similar but not identical mechanisms. Deprivation reduction tended to decrease ingestion by reducing the rate of drinking and size of the first meal. Bombesin, by contrast, reduced the total duration of drinking and the number of meals taken, but had no effect on lick rate. Cumulative intake records suggested that bombesin reduces the volume threshold for termination of drinking such that intake continues at a normal rate below this threshold but ceases above it. This bombesin-imposed threshold for cessation of drinking was between about 5 and 7 ml for individual animals under both food-deprived and nondeprived testing conditions.

Neurotensin-induced hypothermia prevents hippocampal neuronal damage and increased locomotor activity in ischemic gerbils.

Hypothermia induced by surface cooling has shown to protect vulnerable regions of the brain during an ischemic insult. This study evaluated the neuroprotective efficacy of neurotensin, a potent hypothermic agent, using a 5-min carotid occlusion procedure in the gerbil. In Experiment 1, the dose-response and time course of neurotensin-induced hypothermia were evaluated (n = 5/dose). Central infusion of 10, 20, and 30 micrograms neurotensin were found to significantly decrease core body temperature of conscious gerbils within 30 min of administration. In Experiment 2, gerbils pretreated with 30 micrograms neurotensin were permitted to become hypothermic or were maintained at 37 degrees-38 degrees C (rectal) during ischemic insult. Other gerbils were pretreated with peptide vehicle prior to ischemic insult (at 37 degrees -38 degrees C) or underwent a sham procedure (n = 6/condition). At 24 h after surgery, gerbils were tested for increased locomotor activity in an open-field apparatus. Gerbils pretreated with peptide vehicle or neurotensin and maintained at 37 degrees-38 degrees C during ischemia had significantly higher activity levels compared to the other treated groups. In contrast, gerbils made hypothermic with neurotensin exhibited activity levels similar to sham gerbils. Histological assessment revealed that neurotensin-induced hypothermia protected the CA1 region from ischemic damage.

Bombesin-induced hypothermia: a dose-response and receptor antagonist study.

Bombesin infusion into the preoptic area (POA) has previously been shown to induce hypothermia in rats that are food deprived or made hypoglycemic with insulin. The present study evaluated the potency and receptor specificity of this response. Bombesin was microinfused into the POA of food-deprived rats (n = 7) and insulin-pretreated rats (n = 7) at doses of 0, 5, 12, 25, and 50 ng/0.5 microliters. Changes in core body temperature (rectal) were assessed at 1 h. Hypothermia was observed under both conditions with doses as low as 5 ng (3.1 pM) as compared to vehicle (0 ng). In a separate study, infusion of the reduced peptide bond analog (Psi13,14 Leu14)bombesin (2.5 micrograms) prior to bombesin injection (25 ng) was found to prevent the hypothermic response observed in the bombesin control condition. These data suggest that bombesin is a potent hypothermic agent that interacts with gastrin-releasing peptide receptors localized within the POA region to impact thermoregulation.

Bombesin-induced hypothermia and hypophagia are associated with plasma metabolic fuel alterations in the rat.

Microinfusion of bombesin into the preoptic area (POA) has previously been shown to reduce core body temperature and feeding in rats that are food-deprived or made hypoglycemic with insulin. The present study determined the metabolic fuel state of rats under these experimental conditions. In addition, changes in plasma metabolic fuels following the microinfusion of bombesin (50 ng/0.25 microliters) into the POA were evaluated. Rats (n = 8) were tested under conditions of food satiation, food deprivation (20 h), and insulin pretreatment (10 U/kg). Prior to peptide infusion, food-deprived rats exhibited the expected elevation in free fatty acids coupled with a small decline in plasma glucose. Insulin treatment resulted in hypoglycemia which persisted for at least 120 min. Following bombesin infusion, free fatty acids and corticosterone levels were elevated in food-sated rats. Food-deprived rats exhibited elevation in plasma glucose, free fatty acids, and corticosterone following peptide infusion. In insulin-treated rats, bombesin attenuated the hypoglycemia observed in controls and increased corticosterone levels. These findings suggest that bombesin-like peptides localized within the POA may participate in the regulation of metabolic fuels.

Bombesin produces hypothermia in rats pretreated with 2-deoxy-D-glucose.

Previous research has shown that microinfusion of bombesin into the preoptic area (POA) decreases core body temperature in rats that are food-deprived or made hypoglycemic with insulin. The present study employed 2-deoxy-D-glucose, a competitive inhibitor of glycolysis, to further investigate the importance of a reduction in glucose utilization in the production of bombesin-induced hypothermia. Rats (n = 7) were pretreated with 2-DG (0, 25, 50, 100, 200 mg/kg; IP) followed by bombesin (100 ng/1.0 microliters) microinfusions into the POA. The highest dose of 2-DG (200 mg) was also tested in the absence of bombesin as a control. Pretreatment with 2-DG resulted in a dose-related reduction in Tb following bombesin. Injections of 2-DG alone did not significantly alter Tb. The results provide additional evidence that the production of bombesin-induced hypothermia in fasted rats is linked to a reduction in glucose utilization.

Refeeding attenuates bombesin-induced hypothermia in the rat.

The tetradecapeptide bombesin is a potent agent in producing hypothermia when injected centrally. Bombesin-induced hypothermia at normal ambient temperature occurs under conditions of food deprivation or insulin-induced hypoglycemia. This experiment examined the effect of refeeding on the duration of bombesin-induced hypothermia. Rats (n = 7) received microinfusions of bombesin (0.1 microgram/1.0 microliter) into the preoptic area under separate conditions of food deprivation (18 h) and insulin pretreatment (10 U/kg, IM). Core body temperature was evaluated over a period of 4 h with or without food available during testing. Hypothermia was observed under all conditions during the first 2 h. Food-deprived and insulin-pretreated rats not permitted access to food remained hypothermic until at least 4 h following bombesin. These results are discussed in terms of the possible role of glucose availability in the production and duration of bombesin-induced hypothermia.

Microinfusion of bombesin into the hypothalamic paraventricular nucleus produces hypothermia in the insulin-pretreated rat.

Bombesin-like peptides are widely distributed in the mammalian central nervous system and participate in the regulation of a variety of autonomic functions. Central injection of bombesin produces hypothermia at normal ambient temperatures, but only if the rat has been food-deprived or made hypoglycemic with insulin. Two experiments were conducted to reevaluate the impact of bombesin microinfusion into the hypothalamic paraventricular nucleus (PVN) on core body temperature and feeding behavior. In Experiment 1, bombesin (0.05 and 0.1 microgram/1.0 microliter) produced hypothermia, but not hypophagia, in rats (n = 5) pretreated with insulin (10 U/kg; IM). Since a similar response was observed in rats with injection sites adjacent to the PVN, a smaller injection volume was evaluated in Experiment 2. Hypothermia, but not hypophagia, was observed in rats (n = 5) pretreated with insulin following bombesin (0.025 and 0.05 micrograms/0.5 microliter). Bombesin did not produce hypothermia in rats with injection sites outside of the PVN. These findings suggest that the PVN is a sensitive site for bombesin-induced hypothermia.

Bombesin-induced hypothermia in the insulin-treated rat: effect on tail-skin temperature.

Bombesin-like peptides are widely distributed in the mammalian central nervous system and appear to participate in the regulation of a variety of autonomic functions. Bombesin has been shown to alter feeding behavior, locomotor activity, and thermoregulation. Microinfusion of bombesin into the preoptic area of the hypothalamus produces a reduction in core body temperature, but only if the rat has been cold-exposed, food-deprived, or pretreated with insulin. The mechanism for bombesin-induced hypothermia under the latter two conditions is unknown. The present study evaluated the possible contribution of peripheral heat loss mechanisms in bombesin-induced hypothermia. Rats were administered insulin (10U/kg, Regular Iletin I i.m.) or saline followed by an intrahypothalamic injection of bombesin (.05 microgram/.25 microliter) or peptide vehicle. Rectal and tail-skin temperatures were measured continuously for 120 min. Changes in temperature were evaluated at 30, 60, 90, and 120 min., using analysis of variance. As previously demonstrated, bombesin produced hypothermia in rats pretreated with insulin. This reduction in core temperature was not associated with any significant alteration in tail-skin temperature. Results suggest that bombesin-induced hypothermia in rats pretreated with insulin may not be mediated by an increase in peripheral heat loss.

Bombesin-induced hypothermia in food-deprived rats.

Three separate experiments were conducted to characterize the effects of bombesin on core body temperature in food-deprived rats and further evaluate the role of food deprivation in this response. Previous research has demonstrated that naloxone reverses bombesin-induced hypothermia in cold-exposed rats. The present study was unable to demonstrate a similar reversal under conditions of food deprivation following naloxone. In a second experiment, cold exposure (11 degrees C) was shown to potentiate bombesin-induced hypothermia in food-deprived rats. Taken together, these data support the notion that bombesin-induced hypothermia in food-deprived and cold-exposed rats represent the activation or disruption of different thermoregulatory mechanism(s). The final experiment evaluated the effects of bombesin microinfusion into the preoptic area of the anterior hypothalamus on core body temperature. Animals were tested under conditions of food satiation, food deprivation, and insulin pretreatment. Bombesin produced hypothermia in food-deprived and insulin-treated rats. No hypothermia was observed in food-satiated rats. Our finding supports the notion that factors associated with the fasting state are important for the production of hypothermia by bombesin in food-deprived rats.

Effects of site-specific CNS microinjection of cholecystokinin on lordosis behavior in the male rat.

We have previously demonstrated that intracerebroventricular injections of sulphated cholecystokinin octapeptide (sCCK-8) had a dramatic facilitatory effect on lordosis behavior in the gonadectomized, estrogen-primed male rat. In the female, sCCK-8 facilitates or inhibits lordosis when microinjected into the medial preoptic nucleus (MPN) or ventromedial nucleus of the hypothalamus (VMH), respectively. In order to identify sCCK-8 responsive sites that modulate lordosis behavior in gonadectomized males, sCCK-8 was microinjected into the MPN or VMH. Sulphated CCK-8 significantly increased lordosis behavior when microinjected into the MPN of estrogen-primed males, but had no significant effects when microinjected into the VMH. These results imply that CCK-sensitive neural substrates within the MPN may act to disinhibit lordosis in the gonadectomized, estrogen-primed male rat. The lack of an effect of VMH injection of sCCK-8 on lordosis in males is discussed in terms of possible sex differences in sCCK-8-sensitive lordosis-modulating circuits.

Bombesin produces hypothermia in insulin treated rats.

Previous studies have shown that central injection of bombesin produces hypothermia in food-deprived, but not food satiated rats at normal ambient temperatures. The present study evaluated the effects of bombesin on core body temperature (Tb) and feeding behavior in rats pretreated with insulin. Administration of bombesin (0.25, 0.5, and 1.0 microgram) into the lateral cerebral ventricle produced hypothermia in rats injected with insulin (10 U/kg; i.m.). No significant change in core temperature was observed in control rats following bombesin. Insulin treatments significantly stimulated feeding behavior and the highest dose of bombesin significantly reduced feeding behavior. The results demonstrate bombesin-induced hypothermia under metabolic conditions similar to acute starvation. These findings are consistent with the hypothesis that bombesin-induced hypothermia in food-deprived rats is directly related to the fasting state.

Injections of cholecystokinin into the ventromedial hypothalamic nucleus inhibit lordosis behavior in the rat.

In the central nervous system, the ventromedial nucleus of the hypothalamus (VMH) is a primary locus for the induction of lordosis behavior by estrogen. Tissue levels of cholecystokinin and its binding sites in the VMH are modulated by estrogen, suggesting a role for cholecystokinin in the regulation of lordosis behavior. The effects of exogenous cholecystokinin octapeptide (CCK-8) were examined by injecting CCK-8 into the VMH and measuring the frequency of lordotic responses. CCK-8 inhibited the frequency of lordotic responses in a dose-related fashion when the control rats had lordosis quotients of 60 and above. This inhibition is in agreement with previously reported results after peripheral injection of CCK-8, and is consistent with the hypothesis that CCK-8 in the VMH in involved in the regulation of lordosis behavior in the estrogen-primed rat.

Effects of cholecystokinin on male copulatory behavior and lordosis behavior in male rats.

Because the distribution of cholecystokinin octapeptide (CCK-8) within the hypothalamus and limbic system overlaps with steroid concentrating regions, and because these areas are involved in the regulation of reproductive behaviors, we examined the effects of exogenous CCK-8 on male copulatory behavior and lordosis behavior in the male rat. Peripheral administration of a dose of CCK-8 that altered lordosis behavior in females (3 micrograms/kg, intraperitoneal) was ineffective in altering male copulatory behavior in males, either before or after gonadectomy, and was also ineffective in altering lordosis behavior after estrogen priming. In a separate experiment, CCK-8 injected into the lateral ventricle also did not affect male copulatory behavior, but lordosis behavior was increased dramatically after gonadectomy and estrogen priming. Although these results do not answer the question whether CCK-8 is acting to inhibit a neural system that normally suppresses lordosis behavior or is acting to stimulate a facilitatory circuit, these results do indicate the existence of an estrogen sensitive neural substrate in males on which CCK can act to facilitate lordosis behavior.