A practical method for simultaneous multiple intracerebral implantations for microdialysis in rats.

Many experimental designs require the chronic implantation of different elements destined to act as channels that facilitate the information conveyance between the brain and some external devices or vice versa. Electrodes for electrophysiological or electrochemical recording or brain stimulation, and guide shafts for drug administration or chemical monitoring of the extracellular space are the most common examples of channels serving those purposes. The stereotaxic implantation of one or more of those experimental tools in the same antero-posterior plane is relatively easy, but surgery is nonetheless more complicated when two or more elements have to be placed using totally different coordinates. In those cases the current strategy consists in the successive implantation of the elements, waiting for the hardening of the dental acrylic destined to fix one of them in place before dealing with the next. This procedure takes time, is considerably more laborious than surgery for single elements and is particularly difficult when the elements have to be implanted in close proximity. The present report describes a method that simplifies surgery for multiple intracerebral implantation and allows the simultaneous and exact placement of as many electrodes or guide shafts as is practical in any experimental design. The method requires the previous construction of a jig or template designed to temporarily hold the elements to be implanted, allowing them to assume and keep the same positional relationship that they should have when definitively in place within the skull. The design may vary according to the type of elements to be implanted and the coordinates required for each particular experiment, but here it is illustrated describing the assembly of a particular jig for the simultaneous implantation of guide shafts for ulterior microdialysis in the prefrontal cortex (PFC), nucleus accumbens (NAC) and striatum (STR). Some rules can be derived from this particular case to make the method a more general one and suitable for any combination of elements and stereotaxic coordinates.

A multipurpose four-channel fluid swivel.

This article describes the construction of a functional quadruple fluid swivel with low propensity to leaking and low resistance to rotation. It was obtained through the addition of a fourth channel and some modifications performed on the rotary unit of a triple channel fluid swivel recently described. Those modifications included alterations of the Touhey-Borst adapter (Becton-Dickinson), and the development of a new method of fixation of the rotary tubes to the rotary unit of the swivel using nuts and screws. With those improvements the former swivel was transformed in a still more versatile device that can be used in experimental designs requiring the use of this relatively high number of channels. It can be used, for instance, in simultaneous off-line brain microdialysis of up to four different areas or on-line brain microdialysis in two areas; in push-pull perfusions of two brain regions; in studies determining the neuropharmacokinetics and pharmacodynamics of any compound and combining continuous drug infusion through one channel, double brain microdialysis to describe the distribution characteristics of the drug into the central nervous system as well as to monitor the neurochemical modifications induced by the drug, and continuous plasma sampling to know the plasma pharmacokinetics of the drug. These and several other methods used in neuroscience research can also be combined using the swivel described here.

Extracellular glutamate increases in the lateral hypothalamus and decreases in the nucleus accumbens during feeding.

Glutamate release was monitored in the lateral hypothalamus and the nucleus accumbens during a meal using 30 s resolution microdialysis and capillary zone electrophoresis with laser-induced fluorescence. A significant increase in hypothalamic glutamate and a decrease in accumbens glutamate were observed. These results, added to previous pharmacological studies, suggest that glutamatergic synapses in the lateral hypothalamus and the nucleus accumbens might be involved in the control of feeding behavior.

Changes in dopamine and acetylcholine release in the rat lateral hypothalamus during deprivation-induced drinking.

Neurochemical changes in the rat lateral hypothalamus during drinking were assessed in 20 min sampling intervals, using in vivo brain microdialysis. Water-deprived animals drank (11 +/- 1 ml) during the hour that water was available. Drinking was maximal (7.8 +/- 0.7 ml) during the first 20 min after water presentation and minimal during the last 20 min (0.5 +/- 0.4 ml). There was a local enhancement in DA turnover evidenced by an increase in the extracellular levels of dopamine (DA) (155 +/- 47% during the second sample after water presentation as compared to predrinking levels) and dihydroxyphenyl acetic acid (DOPAC) (132 +/- 9.7% in the sample that followed water removal). There was also an initial increase in the acetylcholine (ACh) release (145.1 +/- 21.7%) during the first 20 min after water presentation followed by a reduction (50.12 +/- 18%) 20 min later. These changes are congruous with previously published results suggesting that both neurochemical systems are involved in the regulation of water intake. Considering that the exogenous administration of cholinergic drugs in this hypothalamic area elicits drinking, the initial increase in ACh release could be interpreted as one of the neurochemical events driving this behavior. Since the local blockade of D2 receptors has been shown to result in drinking the progressive increase in DA turnover detected in this study, as well as the concomitant reduction in ACh release, could be involved in drinking attenuation.

Selective action of acute systemic clozapine on acetylcholine release in the rat prefrontal cortex by reference to the nucleus accumbens and striatum.

The effects of i.p. clozapine [0 (n = 6), 5 (n = 5), 10 (n = 5), 20 (n = 9) and 40 (n = 5) mg/kg] on acetylcholine (ACh) release in the prefrontal cortex (PFC), nucleus accumbens (NAC) and striatum (STR) were studied by simultaneous triple microdialysis in freely moving rats. Clozapine dose-responsively increased extracellular ACh in the studied areas. The effect was larger in the PFC. Comparisons of the slopes of the regression equations showed differences between the effects in PFC and nucleus accumbens (t = 4.29; df = 56; P < .001) and PFC and STR (t = 4.56), but not between nucleus accumbens and STR. These differential actions were not artifacts of the simultaneous perfusion because clozapine (20 mg/kg) increased ACh levels during single microdialysis of the PFC (353 +/- 72%; n = 5) or STR (168 +/- 24%; n = 5), in the same proportion as the respective increases in those areas during the simultaneous triple microdialysis (PFC = 330 +/- 41%; STR = 144 +/- 18%; n = 9). Local infusion of tetrodotoxin (10 microM) reduced ACh in the areas studied to about 30% of the mean baselines, confirming the neuronal origin of this neurotransmitter. Extrapolation of these results to humans suggests that adequate levels of cholinergic activity in the PFC are required for mental health, and that a similar ACh release in the human PFC by clozapine could be therapeutic. The low impact on striatal ACh could explain the lack of extrapyramidal symptoms by clozapine.

Atropine decreases drinking but not feeding and induces less hypothalamic acetylcholine release in diabetic rats.

Drinking, feeding and hypothalamic extracellular acetylcholine (ACh) release was measured before and after the administration of several doses of atropine sulfate in streptozotocin (STZ)-diabetic and normal rats. Drinking but not feeding was dose-relatedly decreased by i.p. or intrahypothalamic injections of atropine in STZ-diabetic rats. Hypothalamic ACh release, as measured by microdialysis, increased less (dose-related) in diabetic than normal rats following an i.p. administration of atropine. Ach basal levels were the same in both groups. These results are discussed in terms of a hyperactive hypothalamic cholinergic (muscarinic) system involved in the diabetic polydipsia.

Systemic and local cocaine increase extracellular serotonin in the nucleus accumbens.

The effect of systemic or intra-accumbens injections of cocaine on serotonin (5-HT) overflow was studied by nucleus accumbens microdialysis in freely moving rats. In Experiment 1, cocaine was injected intraperitoneally at 0, 10, 20, and 30 mg/kg. In Experiment 2, cocaine (3.6, 7.2, and 14.4 mM), lidocaine (7.2 mM), or saline was infused through the probe by reverse microdialysis. Extracellular serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were measured by high-pressure liquid chromatography and electrochemical detection. Systemic administration of cocaine induced a dose-related increase in 5-HT overflow and a decrease of 5-HIAA. Intra-accumbens cocaine infusion also caused a dose-related increase in 5-HT, but no effect on 5-HIAA. As a control for local anesthesia, equimolar lidocaine did not increase 5-HT. The difference between lidocaine and cocaine was not due to unequal diffusion out of the probe, because previous in vivo calibration of the probe showed that more lidocaine than cocaine diffused out of the probe when equimolar solutions were infused. These experiments suggest that systemic cocaine acts on the nucleus accumbens to increase synaptic 5-HT.

Diabetes decreases limbic extracellular dopamine in rats.

Mesolimbic dopamine (DA) was measured by ventral striatum (including nucleus accumbens) microdialysis in freely moving streptozotocin (STZ)-diabetic male rats. DA and dihydroxyphenylacetic acid (DOPAC) basal levels and amphetamine-induced DA increase were lower in diabetic than in normal rats. These results are discussed in terms of decreased DA neuron activity and DA receptor hypersensitivity in the mesolimbic system of STZ-diabetic rats.

Testosterone modulates mesolimbic dopaminergic activity in male rats.

Male rats were castrated before puberty. When they were adult, the activity of their mesolimbic dopamine system was tested by ventral striatum microdialysis. Amphetamine injections increased dopamine more in castrated rats than in normal rats. This exaggerated response was attenuated by testosterone replacement therapy. The mechanism by which androgens modulate the activity of the mesolimbic dopamine system is discussed.

Dopamine increase in the prefrontal cortex correlates with reversal of haloperidol-induced catalepsy in rats.

The mechanism by which forced swimming reverses, haloperidol-induced catalepsy was examined by measuring dopamine (DA) turnover in the nucleus accumbens-ventromedial caudate (NAC-C) and the prefrontal cortex (PFC) in rats. DA and its metabolites 3,4-dihydroxiphenylacetic acid (DOPAC) and homovanillic acid (HVA) were assessed by microdialysis and high pressure liquid chromatography with electrochemical detection (HPLC-ED) after systemic administration of a cataleptic dose of haloperidol (5 mg/kg) or saline. Haloperidol-induced catalepsy was temporarily suppressed by forced swimming. Haloperidol-treated rats showed an increase of DA, DOPAC, and HVA overflow in the PFC and the NAC-C. This increase was greater in the PFC of rats that were forced to swim. Rats that were not treated with haloperidol but were forced to swim (control group) showed an increase of DA, DOPAC, and HVA in the PFC but not in the NAC-C. Zero micrograms, 5 micrograms, 10 micrograms, and 20 micrograms of DA was bilaterally injected in the PFC of cataleptic rats to evaluate the hypothesis that DA in the PFC reverses catalepsy. Haloperidol-induced catalepsy was diminished by bilateral microinjections of 10 micrograms and 20 micrograms but not by 5 micrograms of DA in the PFC. The higher the dose of DA, the longer the decrease of catalepsy. These results suggest that an increase of DA turnover in the PFC might mediate temporal suppression of haloperidol-induced catalepsy. The mechanism by which the mesocortical DA system reduces catalepsy is discussed.

Colinear laser-induced fluorescence detector for capillary electrophoresis. Analysis of glutamic acid in brain dialysates.

Experiments with capillary electrophoresis using a laser-induced fluorescence detector with a colinear optical arrangement demonstrated several important points. First, increasing the numerical aperture of the microscope objective that is used simultaneously for focusing the excitation laser light as well as collection of emitted fluorescence enhances the signal used for the measurement of the emitted fluorescence and at the same time decreases the noise of interfering light. Second, detection of fluorescein-labelled amphetamine was performed at high-picomolar (10(-10) M) levels. Third, the signal-to-noise ratio of 280 found at the above-mentioned picomolar concentrations indicates that the measurement of low-picomolar concentrations (10(-12) M) of this compound in biological samples should be possible. Fourth, narrow-bore capillaries (5-10 microm internal diameter) were used to detect the neurotransmitters glutamic acid and aspartic acid as their naphthalene-2,3-dicarboxaldehyde derivatives in brain dialysates obtained from a freely moving rat. A mathematical model was developed to explain the relationship between numerical aperture, working distance, magnification of the lens, noise due to laser scattering and signal due to fluorescence. The model correctly predicted the observed values of photomultiplier tube current due to both laser scattering and fluorescence. The potential of the application of capillary electrophoresis with laser-induced fluorescence detection in the neurosciences is discussed.

Ventromedial hypothalamus vs. lateral hypothalamic D2 satiety receptors in the body weight increase induced by systemic sulpiride.

Two experiments were conducted in order to see if dopamine satiety receptors in the lateral hypothalamus or satiety mechanisms in the ventromedial hypothalamus were involved in the hyperphagia and body weight increase induced by systemic sulpiride. In the first experiment, it was shown that systemic sulpiride (20 mg/kg) does not block the anorexia caused by intraperifornical injections of amphetamine. In the second experiment, sulpiride (20 mg/kg during 18 days) did not produce an additional increase in body weight in previously VMH-lesioned female rats. This last fact cannot be explained by a ceiling effect since insulin (5 U/day during 7 days) increased body weight in the same VMH rats in which sulpiride was not effective. These results do not support the hypothesis that systemic sulpiride reaches the perifornical dopamine D2 receptors to disinhibit feeding, but suggest instead an involvement of the ventromedial hypothalamus. This last suggestion is more in agreement with the hypothesis that sulpiride alters feeding and body weight gain through the induction of a functional gonadectomy.

Hypothalamic sites affecting masticatory neurons in rats.

The effects of hypothalamic electrical stimulation and dl-sulpiride injections on the unit activity of masticatory trigeminal neurons were assessed in rats. Unilateral electrodes or bilateral cannulas were implanted in the perifornical hypothalamus. The animals which exhibited eating to electrical stimulation or to sulpiride injections were selected. Then, under urethane anesthesia, electrical stimuli or sulpiride injections were applied at sites which elicited eating while a passive jaw movement-related neuron was recorded. Electrical stimulation or sulpiride injections affected the basal firing rate of 15 out of 32 (53%), and 13 out of 17 (76%) jaw movement-related neurons, respectively. The basal firing rate of 3 out of 18 (16.6%) was affected by electrical stimulation of noneliciting feeding nearby places. Facilitation was observed in 7 out of those 13 jaw movement-related trigeminal neurons after intrahypothalamic sulpiride injections. When dopamine was injected in the hypothalamus 3 minutes before sulpiride, this drug could only affect the basal firing rate of 1 out of 12 (8%) jaw movement-related neurons. The electrical stimulation and sulpiride injections into the hypothalamus had the same inhibitory or excitatory effect on a given trigeminal neuron as the passive jaw movement did. A similar phenomenon was observed with the sulpiride-induced facilitation. These results suggest that hypothalamic D2 satiety receptors modulate brain stem feeding reflexes.

Mechanism of the sex-dependent effect of lithium on body weight in rats.

Two experiments are reported here. First, the effect of lithium chloride (1, 2 and 4 mEq/kg IP for 21 days) on body weight was assessed in female and male rats. Food intake was measured in the rats treated with 2 mEq/kg. All the doses tested significantly increased body weight in female rats. A linear relationship between body weight gain and lithium dose was also observed. In contrast, in male rats, the low doses of lithium (1 and 2 mEq/kg) did not affect body weight, whereas the high dose (4 mEq/kg) decreased body weight. These results confirm previous reports on a sex-dependent effect of lithium on body weight in rats. In the second experiment, body weight and food intake were assessed in female rats treated with lithium alone, or in combination with insulin or sulpiride, a D2 dopamine receptor blocker. It was found that the effect of lithium on body weight and feeding was additive to the effects of sulpiride and insulin. These findings are indirect evidence that lithium enhances body weight in rats by a different mechanism than the one described for sulpiride or insulin.

Puberty modifies sulpiride effects on body weight in rats.

Sulpiride (20 mg/kg, i.p.) for 21 days increased body weight in prepubertal, peripubertal and adult female rats. The increment was higher in the adult group, where a significant hyperphagia was also noted. In males, the same treatment tended to decrease body weight in the peripubertal and adult groups while in the prepubertal animals an increase of body weight without a significant hyperphagia was observed. These results are discussed in terms of an hypothetical sulpiride-induced reduction in serum gonadal steroid levels.

Sensory-specific satiety: food-specific reduction in responsiveness of ventral forebrain neurons after feeding in the monkey.

It has been shown previously that some neurons in the lateral hypothalamus and substantia innominata respond to the sight of food, others to the taste of food, and others to the sight or taste of food, in the hungry monkey. It is shown here that feeding to satiety decreases the responses of hypothalamic neurons to the sight and/or taste of a food on which the monkey has been satiated, but leaves the responses of the same neurons to other foods on which the monkey has not been satiated relatively unchanged. This suggests that the responses of these neurons in the ventral forebrain are related to sensory-specific satiety, an important phenomenon which regulates food intake. In sensory-specific satiety, the pleasantness of the sight or taste of a food becomes less after it is eaten to satiety, whereas the pleasantness of the sight or taste of other foods which have not been eaten is much less changed; correspondingly, food intake is greater if foods which have not already been eaten to satiety are offered.

Lateral hypothalamic sites eliciting eating affect medullary taste neurons in rats.

A hypothetical functional relationship between the feeding area of the lateral hypothalamus and the medullary and pontine taste areas was assessed in rats. Multiple electrodes were implanted in the lateral hypothalamus and the animals which exhibited eating upon electrical stimulation were selected. Then under pentobarbital anesthesia, the hypothalamus was electrically stimulated while unit activity of medullary and pontine taste areas was recorded. Electrical stimulation of the hypothalamus at sites which elicited eating affected 22% of gustatory neurons, 13% of proprioceptive neurons sensitive to oropharyngeal stimuli but only 0.2% of the remaining nongustatory neurons. Electrical stimulation at nonfeeding sites affected 3% of the gustatory neurons. The electrical stimulation of the hypothalamus had the same inhibitory or excitatory effect on a given gustatory neuron as the sapid stimulation of the tongue did. These results are discussed in terms of the taste pathways and their possible role in electrically elicited feeding.