A new triple-channel swivel for fluid delivery in the range of intracranial (10 nl) and intravenous (100 microliters) self-administration volumes and also suitable for microdialysis.

This paper describes a new low-torque, bubble-free and multiple-channel swivel of easy construction. This swivel combined with the use of fused silica capillary tubing to connect syringes and injectors, as we recently proposed, allow the accurate and repeated microinjection of low nanoliter volumes (10 nl) in freely moving rats, as required in the intracranial self-administration paradigm. Microinjections can be simultaneously performed in 3 different brain regions. Relatively large volumes in the 10-100 microliters range can be repeatedly administered, as in intravenous self-administration, using the traditional connections with polyethylene (PE) tubing. This swivel allows the execution of experiments involving in vivo microdialysis in up to 3 different brain areas. The internal channel has a very low dead space (4 microliters) and can be used to withdraw small liquid samples and perform on-line microdialysis in freely moving animals. This versatility makes the present swivel appropriate for sophisticated experimental designs involving combinations of intracranial, intravenous and/or intragastric self-administration with microdialysis.

A remote insertion technique for intracerebral microinjections in freely moving animals.

This report describes two improvements to the typical double-cannula microinjection technique. (1) Intracerebral microinjections usually require holding the animal during the insertion of an injector through an implanted guide cannula; however, this is not necessary with the technique described. The injector is made of a long piece of fused silica capillary tubing (145 mm outer diameter x 21.2 cm) which is so small and flexible that it slips through a PE-20 tube (20 cm) that guides it into the implanted guide cannula and down to the desired brain site where it stops. (2) Connection to a microliter syringe is usually done with PE tubing which is leaky, expandable and represents a relatively large dead space that makes it difficult to deliver small, accurate volumes. This problem is avoided by making connection to the syringe via another piece of silica glass capillary tubing. Thus both the injector and its connection to the syringe are made of glass. With these modifications the injector can be inserted without touching the animal, and accurate volumes in the low nanoliter range can be delivered.

Triple electrical channels on a triple fluid swivel and its use to monitor intracranial temperature with a thermocouple.

A low-torque, bubble-free and multiple-channel fluid swivel of easy construction was recently described. This paper describes the design, construction and testing of 3 electrical channels added to the original fluid swivel. The new channels were tested monitoring intrahypothalamic temperature (T(hy)) by means of a copper-constantan thermocouple in freely moving rats, before and after a single intraperitoneal (i.p.) amphetamine injection (3 mg/kg). This test showed an increase in T(hy) after the injection and the maintenance of the electrical continuity along the whole testing period, even when the animals were hyperactive. With this improvement the original swivel was transformed in a more versatile device for experiments requiring fluid handlings and electrophysiological manipulations. Electrical stimulation as in kindling or brain self-stimulation, and electrophysiological recordings as in electroencephalography, electromiography, electrocardiography, in vivo voltammetry and even neuronal unit recording, are just examples of the electrophysiological methods that can be combined with drug self-administration and microdialysis using the present device.

Serotonin may play a role in the anorexia induced by amphetamine injections into the lateral hypothalamus.

Amphetamine (AMPH) injections into the lateral hypothalamus (LH) are known to inhibit feeding and this effect has been shown to be mediated by the release and the reuptake blockade of catecholamines. LH serotonin (5-HT) has been suggested to be involved in feeding inhibition and a recent study showed that LH amphetamine infusion increases extracellular dopamine (DA), norepinephrine (NE) and 5-HT, which suggests that 5-HT might also be involved in amphetamine anorexia. The present study investigated this possibility. A correlational study was performed between the anorectic effect of LH unilateral microinjections of each monoamine and the anorectic effect of AMPH. Six groups of male rats were used. The rats in each group were submitted to 2 series of 6 experimental sessions. Each session consisted of one microinjection in 24 h food-deprived rats, followed by the measurement of food intake 30 min later. The first series was similar for all groups and explored the AMPH effect (difference between the mean food intake after 3 AMPH injections (40 micrograms/0.5 microliter) and the mean food intake after 3 saline injections (0.5 microliter)). The second series explored the effects of DA (40 micrograms), NE (25 micrograms), EPI (25 micrograms), 5-HT (25 micrograms) or AMPH again, in a similar way as described for AMPH in the first series. Linear regression analysis on the first series AMPH effect and the amine effect of the 2nd series showed a positive correlation between both series of AMPH, AMPH and DA, and AMPH and 5-HT. This last correlation was replicated in a different group. No correlation was found between AMPH and NE or AMPH and epinephrine (EPI).(ABSTRACT TRUNCATED AT 250 WORDS)

Dipsogenic effect of pituitary adenylate cyclase activating polypeptide (PACAP38) injected into the lateral hypothalamus.

PACAP38 bilaterally injected in the vicinity of the perifornical lateral hypothalamus (pfLH) induced drinking behavior in rats. The animals (n = 12) drank 19.7 +/- 4.1 ml of water during the hour following PACAP38 microinjections (1 nmol/0.5 microliter). In the same rat sulpiride microinjections (45 nmol/0.5 microliter) had relatively mild effects (7.8 +/- 1.4 ml/h). The dipsogenic effects of sulpiride and PACAP38 were well correlated suggesting that both substances trigger drinking behavior activating the same hypothalamic mechanisms. Neither sulpiride nor PACAP38 promoted drinking when injected just 1.3 mm behind the effective zone. This negative result is an evidence of the neuroanatomical specificity of the dipsogenic effects of both substances. These preliminary results suggest that PACAP38 in the pfLH could be a neuropeptide regulating drinking behavior and perhaps body fluid volume and osmolarity and arterial blood pressure.

Sulpiride increases and dopamine decreases intracranial temperature in rats when injected in the lateral hypothalamus: an animal model for the neuroleptic malignant syndrome?

Sulpiride in the perifornical lateral hypothalamus (pfLH) (4, 8 and 16 micrograms/0.5 microliter) increased intracranial temperature (Tic). The hyperthermia started immediately after the injection, peaked 30 min later and lasted for more than 90 min. Sulpiride (12 micrograms) accelerated recovery from hypothermia in anesthetized animals. Forty-five min after sulpiride Tic raised 1.17 +/- 0.06 degrees C. After a control injection the raise was only 0.5 +/- 0.13 degrees C. Locally applied dopamine (DA) (5, 10 and 20 micrograms) 5 min before sulpiride (12 micrograms) attenuated sulpiride hyperthermia. The largest DA dose reduced Tic (-1.21 degrees C) when administered alone. These findings suggest the existence of D2 receptors in the LH involved in thermoregulation. Changes are that D2 receptors in the human LH could be responsible for the neuroleptic malignant syndrome (NMS), and that sulpiride injections in the rat LH could be used as a model for the study of the pathogenesis of this syndrome.

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.

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.

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.

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.

An improved circular tilt-cage shows that intrahypothalamic injections of sulpiride increase locomotion.

A circular tilt-cage was designed and constructed to measure locomotor activity in rats. Some improvements in relation to previously described circular tilt-cages, as the easy calibration and the automation of the data collection, were introduced. The detection system was built interfacing the actimeters through a circuit board to a Casio FP-200 computer. The interface, the computer and the software can be used to count lever presses or other kind of switch closures. This apparatus was used in the present report to quantitate the locomotor activity induced by sulpiride injected in the perifornical region of the lateral hypothalamus. Bilateral injections of sulpiride (8 micrograms/0.5 microliters) increased locomotion immediately after the injections and for the next 75 min. Bilateral amphetamine (20 micrograms/0.5 microliters) did not change activity. Several doses of sulpiride (2, 4, 8, and 16 micrograms/0.5 microliters) increased activity in a dose-dependent fashion. These results are discussed in terms of the existence of postsynaptic D2 receptors in the lateral hypothalamus involved in inhibition of locomotion.

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.

Dopamine in the lateral hypothalamus may be involved in the inhibition of locomotion related to food and water seeking.

Experiments were conducted in male rats to assess the motor effects of bilateral intraperifornical microinjections of sulpiride, dopamine (DA) and other drugs. Sulpiride increased locomotion of the animals in all the experiments reported here. DA (10 micrograms) administered 5 minutes before sulpiride (8 micrograms) reduced the motor stimulant effect of the neuroleptic from 1601.3 +/- 337.6 to 742.5 +/- 180.4 counts/30 min. SCH 23390 (15 micrograms), haloperidol (2.5 micrograms) and atropine (18 micrograms) did not modify the locomotion level of animals acclimated to the actimeters. After carbachol (5 micrograms) the animals attained a level of hyperactivity (1459.5 +/- 146.5 counts/30 min) similar to that induced by sulpiride (1595.7 +/- 365.7 counts/30 min) in the same experiment. In other experiments DA (10 micrograms) administered 30 min before sulpiride again blocked the effect of 8 micrograms of sulpiride, and reduced the initial hyperactivity of food- and water-deprived animals previously familiarized with the actimeters (922.4 +/- 49.38 counts/15 min under saline, vs. 544 +/- 29 counts/15 min under DA). The same DA dose did not modify the initial spontaneous activity of nonfamiliarized nonfood-deprived rats (508.9 +/- 96.1 after saline vs. 520.9 +/- 47.1 after DA). These results suggest the presence of cells in the lateral hypothalamus involved in the control of locomotion. These experiments also suggest that locomotion triggered by the LH may be exploratory behavior essential to the search for water and food. As a corollary, DA in the LH appears to be involved not only in the inhibition of feeding and drinking but also in the inhibition of exploratory and food- and water-directed locomotion.

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.

d-fenfluramine, but not d-norfenfluramine, uses calcium to increase extracellular serotonin.

Microdialysis in the hippocampus of freely moving rats was used to assess extracellular serotonin (5-HT) in response to local infusion of d-fenfluramine and its metabolite d-norfenfluramine with and without local calcium depletion. Verapamil (1 mM) in calcium-free Ringer infused via the microdialysis probe increased extracellular 5-HT and prevented the full increase in extracellular 5-HT normally caused by 1 mM d-fenfluramine. The results suggest d-fenfluramine might act in part as a calcium channel agonist favoring a calcium influx that in turn would trigger the exocytotic process in 5-HT terminals. d-norfenfluramine, on the other hand, was capable of releasing 5-HT, in vivo, in spite of depleted Ca levels.

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.

Sulpiride injections in the lateral hypothalamus induce feeding and drinking in rats.

Amphetamine injections into the lateral hypothalamus inhibit feeding. This effect is blocked by local administration of neuroleptics, suggesting a role for dopamine in feeding inhibition. However, the type of dopamine receptor involved in satiety is not known. Therefore, we tested the effect of intrahypothalamic injections of sulpiride, a specific D2 receptor blocker, on amphetamine anorexia in food-deprived rats, and on spontaneous feeding and drinking in satiated rats. Sulpiride attenuated by 36% the anorexia produced by intrahypothalamic injections of amphetamine. In satiated rats, sulpiride (8 micrograms/0.5 microliter) elicited feeding (mean food intake after sulpiride: 5.4 g, and after vehicle 1.6 g, p less than 0.001), and drinking (mean water intake after sulpiride: 12.3 ml, and after vehicle: 0.9 ml, p less than 0.001). A dose response relationship was found between sulpiride dose and feeding or drinking. Sulpiride-induced drinking was observed in the absence of food, showing that it is not a postprandial phenomenon. These results suggest that hypothalamic D2 receptors might be involved in feeding and drinking regulation.

Rats self-inject a dopamine antagonist in the lateral hypothalamus where it acts to increase extracellular dopamine in the nucleus accumbens.

Local injection of sulpiride to block dopamine (primarily D2-type) receptors in the perifornical lateral hypothalamus (pf-LH) can induce locomotion, feeding, and drinking, and in the present study, local sulpiride induced reward and dopamine (DA) release in the nucleus accumbens. Sulpiride injected bilaterally (4, 8, and 16 micrograms/0.3 microliters), ipsilaterally, or contralaterally (8 micrograms) in the pf-LH increased extracellular levels of DA and its metabolites in the accumbens. Bilateral sulpiride injected posterior and medial to the pf-LH controlled for diffusion to the ventricle or ventral midbrain. Rats self-injected sulpiride (210 ng/21 nl/2 s) in the pf-LH (111 resp/2 h on drug lever vs. 20 resp on a blank lever). Thus, cells in the pf-LH establish connections with mesolimbic DA neurons involved in the behavior reinforcement process. Evidently hypothalamic cells with DA receptors normally inhibit aspects of behavior reinforcement. Disinhibition with hypothalamic sulpiride is reward for self-injection and cause of overeating that can lead to obesity.

Mechanism of the body weight increase induced by systemic sulpiride.

Long-term intraperitoneal administration of sulpiride induced body weight increase in female but not in male rats. The hypothesis that systemic sulpiride causes an endocrine unbalance which in turn causes body weight gain and hyperphagia was tested in four experiments. First, it was shown that even when they are on a high-fat diet male rats do not show body weight gain induced by systemic sulpiride. Second, sulpiride suppressed the estrous cycle. Third, gonadectomy prevented the body weight gain induced by systemic sulpiride in female rats. Fourth, estradiol simultaneously administered with sulpiride prevented the expected sulpiride-induced body weight gain. These results are discussed in terms of an hypothetical functional castration produced by systemic sulpiride. The well known hyperprolactinemia, induced by the pituitary D2 dopamine receptor blockade, might bring about an impairment of the steroidogenesis with subsequent decrease in estrogens level, which in turn might be responsible for the hyperphagia and body weight increase induced by systemic injections of sulpiride.

Dopamine-acetylcholine interaction in the rat lateral hypothalamus in the control of locomotion.

Pharmacological, neurochemical, and behavioral techniques were used to characterize DA-ACh interaction within the lateral hypothalamus (LH) in the context of locomotion, feeding behavior, and reinforcement. In Experiment 1, the muscarinic agonist carbachol injected in the LH increased locomotor activity in proportion to dose. In Experiment 2, the same doses of carbachol proportionately increased exctracellular DA in the nucleus accumbens (Nac) as monitored by brain microdialysis. Dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) also increased. In Experiment 3, LH infusion by reverse microdialysis of the D(2) receptor blocker sulpiride released ACh in the LH in a dose-response manner. This suggested that sulpiride disinhibits ACh release via D(2) receptors in the LH and thereby facilitates behavior. Confirming this in Experiment 4, local LH atropine 5 min before sulpiride suppressed the locomotor response to sulpiride for about 20 min. These results suggest that sulpiride acts in the LH by disinhibiting a hypothalamic locomotor mechanism that is cholinergically driven and connected with the mesoaccumbens dopamine pathway. Given prior results that local sulpiride in the LH can induce hyperphagia and reward, this system may be involved in searching for food and rewarding feeding behavior. In conclusion, DA acts in the LH via D(2) receptors to inhibit cholinergic neurons or terminals that are part of an approach system for eating.