Introduction

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Carbon monoxide (CO) is an air pollutant and one of the most important constituents of cigarette smoke. The primary target for CO toxicity is the central nervous system, as shown by the impairment of ongoing behavior produced by low concentrations of inhaled gas. In particular, the developing brain is extremely vulnerable to chronic, relatively mild, reduction in oxygen availability induced by CO (Annau and Fechter, 1994; Cagiano et al., 1998).

Previous findings have shown that prenatal exposure to CO, at a concentration (150 ppm) below that associated with gross malformations and/or overt neurotoxic effects, induces learning and memory deficits in rat offspring (Mactutus and Fechter, 1984; Di Giovanni et al., 1993). Moreover, recent clinical reports indicate that children born to women who smoked during pregnancy exhibited poorer performance on cognitive tasks, as well as an impaired intellectual development (Frydman, 1996; Fried et al., 1998).

On the basis of these evidences, experiments have been carried out to investigate whether the cognitive deficits produced by a prenatal exposure model simulating the CO exposure observed in human cigarette smokers (Mactutus and Fechter, 1984; Di Giovanni et al., 1993) could be related to alterations in long-term potentiation (LTP), the most intensively studied cellular and molecular model for learning and memory (Collingridge, 1992; Bliss and Collingridge, 1993; Nicoll and Malenka, 1995).

We have conducted an in vitro electrophysiological study in rat offspring (postnatal days 15-30) chronically exposed to 150 ppm of CO during gestation. The synaptic transmission between the Shaffer collateral terminals and hippocampal CA1 neurons, before and after tetanization, was investigated. In particular, we analyzed input/output (I/O) relationship, post-tetanic potentiation (PTP), short-term potentiation (STP), LTP, and paired-pulse facilitation (PPF), a short-lasting form of synaptic plasticity related to Ca²⁺-mediated transmitter release (Katz and Miledi, 1968; Asztely et al., 1996).

CO is an endogenously generated gas that plays an important physiological role in the brain (Verma et al., 1993). In particular, it has been suggested that CO could be involved in the phenomenon of LTP. In fact, LTP maintenance (or expression) requires the increase in synaptic strength mediated by -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)- and metabotropic-glutamate receptor activation (Bashir et al., 1993; Bortolotto and Collingridge, 1993; Bortolotto et al., 1999), as well as by the production of retrograde intercellular messengers, notably nitric oxide (NO) and CO itself (Hawkins et al., 1994; Medina and Izquierdo, 1995). Accordingly, when CO is applied to slices in association with a weak tetanic stimulation, unable per se to produce LTP, it induces a rapid and long-lasting enhancement of synaptic response in the CA1 region of the hippocampus (Zhuo et al., 1993).

Thus, the aim of a second series of experiments was to investigate whether prenatal exposure to a low concentration of CO could affect the activity of two enzymes in the hippocampus, NO synthase (NOS) and heme-oxygenase 2 (HO-2), regulating the production of NO and CO, respectively (Snyder et al., 1998). Because 96% of NOS activity in the hippocampus is due to the neuronal isoform (Huang et al., 1993), we focused our attention on neuronal (n)NOS, although the importance of the endothelial NOS form in LTP formation has been recently stressed (Son et al., 1996; Wilson et al., 1999). Finally, nNOS and HO-2 activities in the frontal cortex and cerebellum were also evaluated.

	Materials and Methods

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Animals and Exposure Conditions. Rats were exposed to CO as previously described (Di Giovanni et al., 1993; Cagiano et al., 1998). Briefly, primiparous Wistar female rats weighing 250 to 280 g were housed in hermetic chambers (Alco Industries, Segrate, Milan, Italy) and exposed to either air (0 ppm of CO, control group) or 150 ppm of CO mixed with air from gestational day 0 (GD 0) to GD 20. Temperature was maintained at 20-22°C and light was on from 8:00 AM to 8:00 PM. Concentrations of CO in each chamber were continuously monitored by an infrared CO detector (CO11 M; Environment SA, Poissy, France) at a wavelength of 4.67 µm. The actual CO concentration deviated by less than 3% from the stated value.

Dam Carboxyhemoglobin (HbCO). Catheters were implanted in the abdominal aorta of separate groups of pregnant CO (0 and 150 ppm)-exposed rats under anesthesia (Equithesin, 3 ml/kg i.p.). Maternal HbCO was measured by a spectrophotometric method described by Rodkey et al. (1979). Briefly, blood samples (10 µl) were taken into a heparinized syringe, diluted about 1000-fold in a solution containing Na₂S₂O₄ (2 mg/ml), and analyzed for their absorbance in the Soret region (390-440 nm) using a UV/visible spectrophotometer (Perkin-Elmer Co., Norwalk, CT). Measurements were performed on GD 10 and 20.

Slices. At postnatal days 15 to 30 (31-95 g b.wt.), transverse hippocampal slices were prepared following standard methods (Mereu et al., 1991; Bortolotto and Collingridge, 1993). Briefly, after deep anesthesia with 4.0% halothane in O₂ was established in rats, they were decapitated, and the brain was rapidly removed under chilled Ringer solution. Slices (350 µm thick) were cut with a vibroslicer (World Precision Instruments, Sarasota, FL) and incubated at room temperature (20 ± 2°C) for at least 60 min and then individually transferred to an interface chamber. Ringer medium contained (mM): NaCl (124), KCl (3.5), NaH₂PO₄ (1.25), NaHCO₃ (22), dextrose (10), MgCl₂ (1), and CaCl₂ (2). The solution was maintained at pH 7.4 by continuous bubbling with 5% CO₂ in O₂.

Electrophysiology. Field excitatory postsynaptic potentials (f-EPSPs) were recorded from stratum radiatum of CA1 pyramidal cells in response to monopolar stimuli (20-µs duration) delivered to the Schaffer collateral/commissural pathway via platinum electrodes. Recording electrodes were filled with the medium (2-4 M). Synaptic responses were sampled at 5 to 10 kHz. Acquisition and analysis were performed by a pCLAMP 5.5/Digidata 1200 system (Axon Instruments Inc., Foster City, CA). The evoked response was measured as the slope of its rising phase after the presynaptic volley. An I/O curve was constructed for each slice by plotting increasing single stimulus intensity [scan (range of increasing currents to construct the I/O curves): 5-100 µA] versus the evoked f-EPSP. This curve was used to assess synaptic excitability and to set the stimulus intensity to obtain a test f-EPSP of about 30 to 40% of the maximal response.

Enzyme Measurements: nNOS Assay. NOS activity was assayed according to the method of Bredt and Snyder (1989) with only minor modifications (Kitamura et al., 1995). Briefly, after rapid dissection, hippocampal frontocortical and cerebellar tissues were immediately frozen on dry ice and then stored at 70°C. Subsequently, tissues were thawed and homogenized (1:20, w/v) with a Teflon-glass homogenizer in a 50 mM Tris-HCl buffer (pH 7.4) containing 0.5 mM EDTA, 0.5 mM EGTA, and 0.1 mM phenylmethylsulfonyl fluoride. The supernatant obtained after centrifugation at 48,000g for 30 min was used as the source for nNOS activity to be measured by the conversion of L-[¹⁴C]arginine to L-[¹⁴C]citrulline (and NO in a equimolar ratio). The cytosolic fraction (40 µl, 50-70 µg of protein) was incubated at 37°C for 15 min with 1 mM NADPH, 0.1 mM BH₄, 2 mM CaCl₂, 1 mM dithiothreitol, and 3 µM L-[¹⁴C]arginine (L-[¹⁴C]arginine, 303 Ci/mol specific activity, obtained from Amersham Corp., Little Chalfont, UK) in a total volume of 120 µl. Reactions were stopped by adding 500 µl of ice-cold 20 mM HEPES buffer (pH 5.5) containing 5 mM EDTA. Thereafter, 500 µl of each sample were applied to chromatography columns containing 1 ml of Dowex AG50W-X8 (H⁺ form, 200-400 mesh) equilibrated with 20 mM HEPES (pH 5.5). The newly synthesized L-[¹⁴C]citrulline was specifically eluted with 1 ml of MilliQ water. Liquid scintillation fluid (formula 989; Dupont-NEN, Zaventem, Belgium) was added to the samples, and radioactivity was counted. NOS activity was expressed as picomoles of citrulline formed per milligram of protein per minute.

Enzyme Measurements: HO-2 Assay. HO-2 activity was measured according to the radioenzymatic microassay method of Laitinen and Juvonen (1995). The frozen hippocampal frontocortical and cerebellar tissues were homogenized (1:4, w/v) by sonication (5 × 1 s) in ice-cold 0.1 M potassium phosphate buffer (pH 7.5) containing 50 µM phenylmethylsulfonyl fluoride. The homogenates were centrifuged at 14,000g for 1 min in Fisher (2 ml) tubes. The supernatant formed was used for assaying HO-2 activity, based on the measurement of [¹⁴C]bilirubin formed (in equimolar ratio with CO) from [¹⁴C]heme. Aliquots (5 µl of the supernatant, approximately 50-70 µg of protein) were incubated at 37°C for 10 min with 2 mM NADPH and [¹⁴C]heme ([¹⁴C]hemin chloride, 117 Ci/mol specific activity, >90% pure, purchased from Prof. S. B. Brown, Department of Biochemistry and Molecular Biology, University of Leeds, UK) plus unlabeled heme (1 + 1) to obtain a final concentration of 21.3 µM, in a final volume of 10 µl. The reaction was stopped by adding 190 µl of potassium phosphate buffer. [¹⁴C]Bilirubin formed was extracted into toluene (1 ml) by vortexing the samples and separating the phases with a 2-min centrifugation at 14,000g. The organic phase (700 µl) was decanted into scintillation minivials, Optifluor (Packard Italy, Milan, Italy) was added, and the radioactivity was measured. The mean extraction efficiency was 14%, and enzyme activity was corrected accordingly. The total/blank ratio was of 50%. The HO-2 activity was expressed as picomoles of bilirubin formed per milligram of protein per minute.

Statistics. Values were expressed as means ± S.E. Blood HbCO levels were analyzed by Student's t test with Bonferroni's correction. Electrophysiological data were evaluated by two-way ANOVA for repeated measures followed by Tukey's honestly significant difference test or Student's t test where appropriate. Significance of the rate of occurrence (see Table 2) was calculated by Fischer's exact test. Biochemical data were analyzed by Student's t test.

Animal Care. The experiments have been conducted in accordance with guidelines released by Italian Ministry of Health (D.L. 116/92), the Declaration of Helsinki, and the "Guide for the Care and Use of Laboratory Animals" as adopted and promulgated by the National Institutes of Health.

	Results

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Reproduction Data. As observed previously (Di Giovanni et al., 1993), prenatal CO exposure did not affect dam weight gain, number of dams giving birth, length of pregnancy, litter size at birth, pup weight gain, or postnatal mortality (data not shown).

Dam HbCO. As shown in Table 1, exposure to CO produced a significant increase in maternal blood HbCO levels on both GD 10 and 20 (P < .0001). Moreover, HbCO levels in control rats were significantly increased on GD 20 with respect to those found on GD 10 (P < .001).

Synaptic Excitability. Changes in basal synaptic excitability were investigated by the analysis of I/O relationship and PPF before tetanization. The number of slices exhibiting PTP was also considered a measure of synaptic excitability.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Prenatal exposure to 150 ppm of CO does not change basal synaptic function at CA3-CA1 synapses of hippocampus. A, I/O relationship of f-EPSP (mean ± S.E.) constructed during the 30 min before tetanus in slices taken from animals prenatally exposed to 0 ppm of CO (, n = 24) or to 150 ppm of CO (, n = 20). Stimulus intensity scanned from 5 to 100 µA. B, bar graph summarizing the actual value of PPF before tetanus in control (n = 24) and CO-slices (n = 20). Bars represent the mean ± S.E. of the ratio between the slope of the second and the first f-EPSP (S2/S1 × 100). There was no significant difference between the two groups.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Representative individual traces showing paired f-EPSP responses and presynaptic volley in a control and CO-slice. Responses were evoked by paired stimuli at 50-ms intervals. Traces were taken at 10 (a), 0 (b), and +30 (c) min from tetanization. Each trace is the average of six consecutive f-EPSPs. Bottom, the three traces are superimposed. Note that at +30 min the trace c in the control slice is still potentiated, whereas in the CO-slice trace c is similar to trace a.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Prenatal CO suppresses LTP maintenance. A, averages (mean ± S.E.) of f-EPSP slopes from control () and CO-exposed () rats. Values of f-EPSP slopes have been normalized to the pretetanus period. Each point represents the average ± S.E. of six consecutive responses taken every 5 min. Values of PTP were 236.11 ± 22.63 and 182.05 ± 20.16% in control and CO-slices, respectively (P < .05; Student's t test). B, duration of LTP. Evoked f-EPSPs were considered potentiated until their slope was 20% with respect to baseline. For the control group, LTP duration was estimated by fitting analysis of the curve in A to calculate the interception point where this curve asymptotically subsided to a value of +20%. It occurred at 226.25 min after tetanus. On the contrary the curve describing the f-EPSP time course in CO-slices returned to +20% of baseline in 24.15 min. Bars represent the means ± S.E. obtained from 24 and 20 slices of control and CO-exposed rats, respectively. *P < .0001 versus controls (Student's t test). C, comparison of STP in isolation from LTP in the two groups. These curves were obtained normalizing the two curves in A at the +25-min value. This value was subtracted from all points in the interval between 15 and +25 min in each curve of A, as described by Schulz and Fitzgibbons (1997). The resulting curves were superimposed to show that the STP is similar in the two groups of slices.

STP and LTP. In control slices, the decay of f-EPSP potentiation, after tetanization, followed the typical biphasic curve which is shown in Fig. 3A. Thus, in agreement with previous studies (Bliss and Collingridge, 1993; Bortolotto and Collingridge, 1993; Schulz and Fitzgibbons, 1997), the f-EPSP slope in control slices showed a first fast decremental phase lasting 15 to 30 min (STP), and then it slowly decayed over, at least, the observation time, i.e., 90 min (which was assumed as the minimum time for LTP maintenance or expression). Fitting analysis was done, and the estimated interception point at which the average curve asymptotically subsided to a value of +20%, with respect to the baseline, was at 226.25 ± 18.84 min after tetanus (Fig. 3B). In 23 of 24 tested slices from control rats, LTP was induced and maintained. In only one case did the f-EPSP slope decay below +20% of the baseline before 60 min. None of the control slices exhibited f-EPSP depression (Table 2).

PPF. To determine whether the complete suppression of LTP maintenance in CO-slices was associated with an impairment of glutamate release from presynaptic terminals, we examined the time course of PPF in association with LTP. Although in individual experiments from both groups, PPF increased or decreased with LTP (Fig. 4, A and B), there was no significant change in average PPF ratios along the time course of f-EPSP potentiation either in control or CO-slices (Fig. 4C). However, there was some tendency for PPF in CO-slices to remain higher than in control slices. As shown in Fig. 4C, the first point of the two curves after tetanization did show a significant reduction (P < .02). This reduction, which appears to be due to the small amount of glutamate-mediated PTP remaining in tetanized cells (Manabe et al., 1993), was, however, similar in both groups.

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Time course of PPF in association with LTP. Behavior of PPF in individual slices from control (A) and CO-exposed (B) rats. C, average of the normalized PPF ratio from control slices () and CO-slices (). Note that, although in individual experiments PPF increased or decreased with LTP (A and B), there was no significant change in the average PPF along the time course of f-EPSP potentiation. However, there was some tendency for PPF in CO-slices to remain higher than in control slices. In C the first point of the two curves after tetanization did show a significant reduction (P < .02; Tukey's honestly significant difference test). This reduction, which appears to be due to the small amount of glutamate-mediated PTP remaining in tetanized cells (Manabe et al., 1993), was, however, similar in both groups. Variability between slices for PPF has been also observed by other authors who have used either young or adult rats (Manabe et al., 1993; Wu and Saggau, 1994; Manabe and Nicoll, 1994; Schulz et al., 1995; Asztely et al., 1996).

nNOS and HO-2 Activities. As depicted in Fig. 5, the prenatal exposure to CO provoked a significant (P < .05), although not impressive (32 and 25%), decrease of nNOS and HO-2 activities, respectively. Conversely, both frontocortical and cerebellar enzyme activities of rats exposed to CO prenatally did not differ from those observed in control animals (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Inhibitory effect of the prenatal exposure to CO on hippocampal nNOS and HO-2 activities at 30 days of age. The enzyme sources (supernatants) were incubated in the presence of [14C]arginine and [14C]heme, respectively. The metabolites [14C]citrulline and [14C]bilirubin formed in equimolar amounts with NO and CO were then measured. Data are means ± S.E. from six experiments performed in triplicate. *P < .05 versus the respective controls (Student's t test).

	Discussion

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The present study demonstrates that hippocampal slices from rats exposed to a low concentration of CO during development exhibited an impaired ability to maintain LTP over time. It occurred in association with a modest, but significant, reduction of PTP and in the absence of any observable difference in baseline synaptic transmission between control and CO-slices. Indeed, I/O function, presynaptic volley, pretetanus PPF, and percentage of slices showing PTP were not affected by the gestational exposure to CO.

These results provide the first evidence that altered cellular mechanisms in rat hippocampus might underlie learning and memory deficits induced by prenatal exposure to CO at a concentration (150 ppm) approaching that experienced by offspring of mothers who smoke during pregnancy.

Indeed, previous studies have shown that prenatal exposure to 150 ppm of CO impairs cognitive functions in rat offspring, without affecting nonassociative or motivational parameters (Mactutus and Fechter, 1984; Di Giovanni et al., 1993). The concentration of CO chosen for these studies is of clinical interest because it produces blood HbCO levels comparable to those maintained by human cigarette smokers, depending on the habit (Coles, 1975). Moreover, our data confirm that HbCO levels tend to be somewhat elevated during gestation, reflecting increased endogenous CO production (Longo, 1976), the physiological significance of which is still unclear.

A body of evidence indicates that LTP in the CA1 region of the hippocampus reflects cellular physiological changes critical to the processes of learning and to the intermediate stages of memory consolidation and retention (Bliss and Collingridge, 1993; Nicoll and Malenka, 1995). Different mechanisms regulate early and late phases of LTP in CA3-CA1 synapses of the hippocampus. Thus, whereas LTP induction requires Ca²⁺ influx via N-methyl-D-aspartate (NMDA)-ionotropic receptors, expression/maintenance appears to be supported by various factors located on both pre- and postsynaptic sites (Bashir et al., 1993; Bliss and Collingridge, 1993; Bortolotto and Collingridge, 1993). These factors include an enhanced glutamate release sustained by the production of retrograde messengers, such as NO and CO itself (Hawkins et al., 1994; Medina and Izquierdo, 1995).

Indeed, intracellular CO production, which is dependent on the enzyme HO-2, has to be constantly elevated in order to maintain LTP. Application of CO, paired with a weak low-frequency stimulation, unable per se to generate LTP, produces a long-lasting enhancement of f-EPSP comparable to that caused by a strong tetanus (Zhuo et al., 1993; Hawkins et al., 1994). The inactivation of HO by zinc protoporphyrin IX blocks LTP induction and reverts previously established LTP (Stevens and Wang, 1993; Hawkins et al., 1994; Medina and Izquierdo, 1995). Moreover, HO activity has been reported to increase in the hippocampus, but not in the neocortex or cerebellum, during step-down inhibitory training (Bernabeu et al., 1995). Furthermore, zinc protoporphyrin IX infusion in the hippocampus, but not in the amygdala, inhibits avoidance learning in rats (Fin et al., 1994). Interestingly, the latter effect is similar to that observed in rat offspring exposed to CO prenatally (Mactutus and Fechter, 1984; Di Giovanni et al., 1993).

The rationale of our study was to combine the above-mentioned evidences to further investigate the effects of CO administered during brain development. The complete and selective suppression of LTP maintenance found in CO-slices, long after the exposure cessation, suggests a persistent and complex mechanism.

The present impairment of LTP expression in the offspring of CO-exposed rats was accompanied by significant decreases (32 and 25%, respectively) in both hippocampal nNOS and HO-2 activities. Postnatal enzyme alterations did not appear to involve additional brain regions. In fact, frontocortical and cerebellar enzyme activities, respectively, did not differ from control counterparts at either 30 or 90 days of age (data not presented), when both HO-2 and nNOS in the hippocampus were still markedly affected. Even though the present electrophysiological results have demonstrated that the prenatal CO exposure causes LTP disruption in the hippocampus, a region primarily involved in learning and memory processes, we cannot rule out the possibility that other brain areas could be affected also. Nevertheless, biochemical data support the hypothesis that alterations in the hippocampus could be responsible, in part, for cognitive deficits produced by prenatal exposure to CO. The involvement of NO (see Son et al., 1996, for references) and CO (Grundemar and Ny, 1997) in the induction of LTP has remained controversial, particularly on the basis of genetic "knockout" mice lacking the genes for HO-2 and NOS (Poss et al., 1995; Son et al., 1996; Wilson et al., 1999). However, the contemporaneous decreases in LTP expression and HO-2 and NOS activities observed in CO-exposed offspring is consistent with the postulated role of both CO and NO in LTP (Hawkins et al., 1994). Irrespective of whether or not it has to deal with LTP, the impairment of both enzymes was not unexpected because of the strict correlation existing between colocalized NOS and HO-2 in neurons (Vincent et al., 1994), where the latter activity is needed as a possible defense against the toxic activity of excess newly formed NO (Ding et al., 1999).

It could be hypothesized that gestational CO-exposure might inactivate HO, possibly via an excess of reaction products. If HO activity is reduced, the sensitivity of metabotropic-glutamate receptor could be consequently decreased. Accordingly, HO antagonists actually block the effect of metabotropic-glutamate receptor stimulation (Glaum and Miller, 1993), which, in turn, is an important factor for LTP maintenance (Bashir et al., 1993; Bortolotto and Collingridge, 1993).

Moreover, the fact that PTP was reduced in slices from CO-exposed rats could be related to a decreased sensitivity of glutamate NMDA and AMPA receptors secondary to long-term hypoxia, as recently suggested by Pichiule et al. (1996).

We have also found that the average post-tetanus PPF did not change during LTP in either control or CO-slices. This datum is consistent with the majority of previous studies that have failed to detect any interaction of PPF with LTP in the CA1 region (Manabe et al., 1993; Manabe and Nicoll, 1994; Wu and Saggau, 1994; Schulz et al., 1995; Asztely et al., 1996).

PPF depends on the increase in presynaptic Ca²⁺-mediated release by the second stimulus (Katz and Miledi, 1968; Wu and Saggau, 1994; Schulz et al., 1995), so that the PPF ratio results inversely related to the probability of transmitter release (Katz and Miledi, 1968; Manabe et al., 1993; Wu and Saggau, 1994; Asztely et al., 1996). Therefore, within the hypothesis that CO production in CO-slices would be inhibited, our inability to detect significant variation in post-tetanus PPF between control and CO-slices implies that the role of CO as retrograde messenger is unrelated to Ca²⁺ influx. This is in line with the indication that CO might enhance glutamate release by activating soluble guanylyl cyclase leading to increased formation of cGMP, which does not require Ca²⁺ influx (Heisler, 1986; Hawkins et al., 1994). More generally, it is possible that the presynaptic mechanism underlying LTP has to be sought downstream of terminal Ca²⁺ influx, as suggested by the results of Wu and Saggau (1994).

In conclusion, our findings suggest that learning and memory deficits induced in rat offspring by prenatal exposure to CO could stem, at least in part, from the disruption of the processes required for long-term synaptic storage, as reflected by the suppression of LTP maintenance, possibly via a negative action on the HO and NOS enzymes.

Because the alterations in hippocampal synaptic transmission have been produced by prenatal exposure to CO levels resulting in maternal blood HbCO concentrations equivalent to those maintained by human cigarette smokers, the present data further point out the large risk that the smoking mother poses for her offspring.