In utero Exposure to Anesthetics Alters Neuronal Migration Pattern in Developing Cerebral Cortex and Causes Postnatal Behavioral Deficits in Rats.

During fetal development, cerebral cortical neurons are generated in the proliferative zone along the ventricles and then migrate to their final positions. To examine the impact of in utero exposure to anesthetics on neuronal migration, we injected pregnant rats with bromodeoxyuridine to label fetal neurons generated at embryonic Day (E) 17 and then randomized these rats to 9 different groups receiving 3 different means of anesthesia (oxygen/control, propofol, isoflurane) for 3 exposure durations (20, 50, 120 min). Histological analysis of brains from 54 pups revealed that significant number of neurons in anesthetized animals failed to acquire their correct cortical position and remained dispersed within inappropriate cortical layers and/or adjacent white matter. Behavioral testing of 86 littermates pointed to abnormalities that correspond to the aberrations in the brain areas that are specifically developing during the E17. In the second set of experiments, fetal brains exposed to isoflurane at E16 had diminished expression of the reelin and glutamic acid decarboxylase 67, proteins critical for neuronal migration. Together, these results call for cautious use of anesthetics during the neuronal migration period in pregnancy and more comprehensive investigation of neurodevelopmental consequences for the fetus and possible consequences later in life.

Nullifying drug-induced sensitization: behavioral and electrophysiological evaluations of dopaminergic and serotonergic ligands in methamphetamine-sensitized rats.

Repeated exposure to methamphetamine produces a persistent enhancement of the acute motor effects of the drug, commonly referred to as behavioral sensitization. Behavioral sensitization involves monoaminergic projections to several forebrain nuclei. We recently revealed that the ventral pallidum (VP) may also be involved. In this study, we sought to establish if treatments with antagonists or partial agonists to monoaminergic receptors could "reverse" methamphetamine-induced behavioral and VP neuronal sensitization. Behavioral sensitization was obtained in rats with five once-daily s.c. injections of 2.5mg/kg methamphetamine, an effect that persisted for at least 60 days. After the development of sensitization, 15 once-daily treatments of mirtazapine (a 5-HT(2/3), alpha(2) and H(1) antagonist), SKF38393 (D(1) partial agonist) or SCH23390 (dopamine D(1) antagonist) nullified indices of motor sensitization as assessed by measuring the motoric response to an acute methamphetamine challenge 30 days after the fifth repeated methamphetamine treatment. VP neurons recorded in vivo from methamphetamine-sensitized rats at the 30-day withdrawal time also showed a robust downward shift in the excitatory responses observed to an acute i.v. methamphetamine challenge in non-sensitized rats. This decreased excitatory effect was reversed by mirtazapine, but not by other antagonists that were tested. These data suggest a potential therapeutic benefit for mirtazapine in the treatment of methamphetamine addiction, and point to a possible role for the VP in the sensitization process to methamphetamine.

Regulation of limbic information outflow by the subthalamic nucleus: excitatory amino acid projections to the ventral pallidum.

The subthalamic nucleus (STN), a component of the basal ganglia motor system, sends an excitatory amino acid (EAA)-containing projection to the ventral pallidum (VP), a major limbic system output region. The VP contains both NMDA and AMPA subtypes of EAA receptors. To characterize the physiology of the subthalamic pathway to the VP, and to determine the influence of EAA receptor subtypes, in vivo intracellular recordings, and in vivo extracellular recordings combined with microiontophoresis, were made from VP neurons in anesthetized rats. Of the intracellularly recorded neurons, 86% responded to STN stimulation, and these displayed EPSPs with an onset of 8.7 msec, consistent with a monosynaptic input. The EPSPs evoked in spontaneously firing neurons were nearly twice the amplitude of those in nonfiring cells (13.1 vs 6.8 mV, respectively). As neurons were depolarized by current injection, the latency for spiking decreased from 24.2 to 14.2 msec, although EPSP latency was unaffected. Eighty-seven percent of the extracellularly recorded VP neurons responded to STN stimulation with a rapid and robust enhancement of spiking; the response onset, like the EPSP onset, equaled 8.7 msec. Firing rate was enhanced by NMDA in 94% of the STN-excited cells, and AMPA increased firing in 94% as well. The NMDA-selective antagonist AP-5 attenuated 67% of the STN-evoked excitatory responses, and the AMPA-selective antagonist CNQX attenuated 52%. Both antagonists attenuated 33% of responses, and 78% were attenuated by at least one. This evidence suggests that a great majority of VP neurons are directly influenced by STN activation and that both NMDA and non-NMDA receptors are involved. Moreover, the VP response to STN stimulation appears to be strongly dependent on the depolarization state of the neuron.

Dopamine receptors in the ventral pallidum regulate circling induced by opioids injected into the ventral pallidum.

Recent reports have indicated that the ventral pallidum receives enkephalinergic and dopaminergic inputs. The present study evaluated the contribution of dopamine (DA) receptors to opioid-mediated locomotor functions of the ventral pallidum. Using circling behavior as a motor index, it was determined that injection of metabolically stable analogs of enkephalin, directly into the ventral pallidum, produced a dose-dependent, naloxone-antagonizable, increase in the number of rats rotating contralateral to the injected side, as well as the rate at which the animals turned. The frequency of the contralateral rotation, induced by intra-ventral pallidal injection of DADL (which closely mimics the endogenous enkephalin peptides, exhibiting a high affinity for the delta receptor with a moderate affinity for mu receptors), increased with intraperitoneal pretreatment with amphetamine, suggesting that enhanced release of catecholamines potentiated effects of opioids in the ventral pallidum. Systemic pretreatment with the D2-preferring antagonist haloperidol, blocked the effects of DAMGO (a mu-specific agonist) but not those mediated by DADL. In contrast, the D1-specific antagonist SCH 23390 was an effective blocker of responses to both opioid peptides. Further evidence for differential consequences of activating delta or mu opioid receptors was provided by systemic pretreatment with a cholinergic antagonist; atropine attenuated responding to DADL but not to DAMGO. To ascertain if DA receptors within the ventral pallidum were sufficient to influence mu-mediated circling, it was determined that intra-ventral pallidal injection of SCH 23390 or sulpiride (a D2 specific antagonist), at concentrations that did not produce motor effects, attenuated responding to subsequent intra-ventral pallidal injections of DAMGO. Thus, the opioid receptors and DA receptors that modify responses to activation of opioid receptors are both located within the ventral pallidum. These studies point to the critical role of DA in the ventral pallidum in motor behavior induced by stimulation of opioid receptors in the ventral pallidum.

Dopamine in the rat ventral pallidum/substantia innominata: biochemical and electrophysiological studies.

Recent anatomical literature suggests that dopaminergic projections ascending from the midbrain terminate within the ventral pallidum/substantia innominata (VP/SI). The present investigation evaluated this possibility using standard biochemical and electrophysiologic approaches. Biochemical studies revealed that dopamine and its major metabolites are present within the rat VP/SI. Concentrations of these compounds were diminished greatly when dopaminergic neurons of the substantia nigra were destroyed. Electrophysiologic studies demonstrated that VP/SI neurons often respond to local applications of dopamine with a decrease in firing rate. These observations support the contention that dopamine regulates neuronal activity within the VP/SI and that cells of origin for at least a portion of this projection lie within the substantia nigra.

Spontaneous unit activity in the globus pallidus following cumulative injections of morphine in phenobarbital- or chloral hydrate-anesthetized rats.

The effect of systemically administered morphine on spontaneous unit activity, recorded from the globus pallidus, was evaluated in this study. A wide range of doses of morphine was administered in order to characterize the influences of morphine on pallidal activity in a dose-related manner. Two separate studies were conducted. In the first study, a semichronic preparation, lightly anesthetized with phenobarbitol, was used. Acutely prepared, chloral hydrate-anesthetized rats were utilized in the second experiment. With phenobarbital, morphine caused a significant reduction in pallidal activity in 75% of the cells recorded, whereas only 45% of the pallidal cells responded similarly to injection of morphine in chloral hydrate-anesthetized animals. However, the dose of morphine required to decrease unit activity was substantially less with chloral hydrate than with phenobarbital anesthesia.

Antagonism of GABAergic transmission within the septum disrupts working/episodic memory in the rat.

Male Sprague-Dawley rats, trained to perform a standard or delayed-non-match-to-sample radial arm maze task, were implanted with a single cannula aimed at the medial septal nucleus. A within-subjects design was utilized to examine the effects of intraseptal administration of the GABAergic antagonist bicuculline on performance of these tasks. Bicuculline (0-0.5 microgram/0.5 microliter) infusion produced dose-dependent impairments when administered prior to performance of a standard radial arm maze task. Post-training infusion of bicuculline (0.-0.25 microgram/0.5 microliter) also induced dose-dependent impairments in the delayed version (4 h) of the task. Further testing indicated that post-training administration of a low dose of bicuculline (0.05 microgram) in the delayed version of the task induced a deficit at a 4-h, but not a 1-h, retention interval. The latter indicates that the impairment varied as a function of bicuculline dose and increasing task difficulty (longer retention intervals). Previous observations indicated that post-training administration of the GABAergic agonist muscimol and the antagonist bicuculline could induce deficits in the performance of the delayed task. The present findings demonstrate that intraseptal bicuculline treatment can disrupt ongoing radial maze performance, as well as the maintenance and/or retrieval of memories necessary for performance of the delayed version of the task. These findings suggest that either activation or blockade of intraseptal GABA receptors is sufficient to disrupt working/episodic memory processes. The role of septum and septohippocampal pathway in working/episodic memory is discussed.

Morphine modulation of GABA- and glutamate-induced changes of ventral pallidal neuronal activity.

Microiontophoresis was used to investigate the influence of morphine on the GABA- and glutamate-evoked responses of ventral pallidal neurons recorded extracellularly from chloral hydrate-anesthetized rats. Of the GABA-sensitive neurons (50 of 69 tested) in the ventral pallidum, all displayed a decreased firing rate when GABA was applied, whereas all of the glutamate-sensitive neurons (29 of 40 tested) increased neuronal activity in the presence of glutamate. The majority of ventral pallidal cells tested (65 of 83) were sensitive to iontophoretically applied morphine, and both increases and decreases in neuronal activity were observed. The ability of morphine to alter the ratio between amino acid-evoked activity ("signal") and spontaneous firing ("noise") was used as an indicator of morphine modulation. A morphine subthreshold ejection current, i.e. one that did not change spontaneous firing rate, and a morphine ejection current that produced approximately 50% of the maximum opioid-induced neuronal response were chosen for this evaluation. When morphine was co-iontophoresed with GABA or glutamate, attenuation of the amino acid signal-to-noise ratio was generally seen, though some potentiations were observed. These changes were independent of the direction of morphine-induced changes in spontaneous firing rate. Both sub- and suprathreshold ejection currents were capable of affecting GABA- and glutamate-evoked responses. These data suggest that morphine is a robust ventral pallidal neuromodulator. As ventral pallidal amino acid activity is important in the integration of sensorimotor information, opioid modulation of amino acid transmission in the ventral pallidum may have a profound effect on this integration.

Activation of dopaminergic neurons modulates ventral pallidal responses evoked by amygdala stimulation.

The ventral pallidum is a basal forebrain region that is thought to integrate cognitive processes with motoric behaviors. These functions are influenced by ventral pallidal inputs, which include projections from the amygdala and the ventral tegmental area/substantia nigra zona compacta. By examining the consequences of this convergence at the neuronal level, the present study indicates that electrical activation of ventral tegmental regions releases dopamine in the ventral pallidum which subsequently modulates pallidal electrophysiological responses evoked by stimulating the amygdala. Stimulation-evoked responses were characterized for extracellular single unit recordings of spontaneously active ventral pallidal neurons from chloral hydrate anesthetized rats. Stimulation of the amygdala evoked short latency (< or = 12 ms; possibly monosynaptic) and/or long latency (> 12 ms; polysynaptic) responses in all ventral pallidal neurons tested. Fifty-nine per cent of the tested neurons responded to ventral tegmental stimulation with short latency inhibition, and these neurons were often sensitive to microiontophoretically applied dopamine. Iontophoresis of dopamine antagonists SCH23390 (a D1 antagonist) or sulpiride (a D2 antagonist) attenuated the ventral tegmental-induced inhibitions. These observations indicate that the evoked responding was the result of endogenously released dopamine, and that D1 and D2 receptors were involved in this effect. Ninety-two per cent of the ventral pallidal neurons that demonstrated short latency responses to amygdala stimulation also exhibited short latency responses to activation of the ventral tegmentum. This suggests that these inputs often converge onto the same pallidal neurons. Amygdala-evoked responses were consistently attenuated by prior stimulation of the ventral tegmentum. Similarly, microiontophoretic ejection of dopamine attenuated amygdala-evoked effects. These results indicate that dopamine modulates amygdala-evoked pallidal responses. Such modulation may contribute to the integrative functions of the ventral pallidum.

Naloxone antagonizes striatally-induced suppression of globus pallidus unit activity.

This study was designed to examine the ability of naloxone to antagonize the inhibition of neurons in the globus pallidus produced by electrical stimulation of the caudate nucleus. In rats anesthetized with chloral hydrate, bipolar stimulating electrodes were placed in the head of the caudate and seven-barrel micropipettes were utilized for recording extracellular activity of globus pallidus cells as well as for microiontophoretic application of experimental drugs. In most globus pallidus cells which were inhibited by caudate stimulation, application of naloxone (at currents which antagonized morphine-elicited depression) attenuated the caudate-induced effect. Naloxone did not antagonize depression of globus pallidus neurons induced by gamma-aminobutyrate. The results indicate that caudate stimulation causes the release of endogenous enkephalins which act to suppress neuronal activity in the globus pallidus. Thus, this study supports biochemical and histochemical studies which provide evidence for an enkephalinergic component in the striatopallidal projection.

Substance P in the ventral pallidum: projection from the ventral striatum, and electrophysiological and behavioral consequences of pallidal substance P.

The ventral pallidum of the basal forebrain contains a high concentration of substance P and receives a massive projection from the nucleus accumbens. The present study was designed to determine whether the accumbens serves as a source for substance P-containing fibers in the ventral pallidum and characterize the function of this tachykinin peptide within the ventral pallidum. By combining in situ hybridization for messenger RNA of the substance P prohormone, beta-preprotachykinin, with Fluoro-Gold retrograde labeling from iontophoretic deposits in the ventral pallidum, a population of substance P-containing neurons was demonstrated in the shell and core components of the nucleus accumbens and the ventromedial striatum. The function of substance P within the ventral pallidum was characterized at the level of the single neuron, and the behaving animal. Electrophysiological assessment revealed that approximately 40% of the 97 ventral pallidal neurons tested were readily excited by microiontophoretic applications of substance P or a metabolically stable agonist analog, DiMeC7 [(pGlu5, MePhe8, MeGly9)-substance P5-11]. Response characteristics were distinguished from glutamate-induced excitations by a slower onset and longer duration of action. Recording sites of tachykinin-sensitive neurons were demonstrated to be located throughout the ventral pallidum and within high densities of fibers exhibiting substance P-like immunoreactivity. When behaving rats received microinjections of DiMeC7 into this same region, the animals displayed an increase in motor activity, with a response threshold of 0.1nmol per hemisphere. These results verify the existence of a substantial substance P-containing projection from the nucleus accumbens to the ventral pallidum. The projection likely serves to excite ventral pallidal neurons for these neurons readily increased firing following local exposure to tachykinins. Furthermore, an increase in motor behavior appears to be a consequence of this neuronal response.

Within-subject decline in delayed-non-match-to-sample radial arm maze performance in aging Sprague-Dawley rats.

A within-subject design was used to examine delayed-non-match-to-sample radial arm maze performance in aging (6-18 months) male Sprague-Dawley rats. A decrease in correct choices and an increase in retroactive errors were observed at all retention intervals at 18 months of age compared with performance at 6 or 12 months. No age by retention interval interaction was observed. Neither age nor increasing retention interval influenced proactive errors during the retention test. The observation of an age- and delay-dependent increase in retroactive errors, but not proactive errors, suggests that the deficit relates to a memory dysfunction as opposed to a generalized performance deficit.

Delayed-non-match-to-sample performance in the radial arm maze: effects of dopaminergic and gabaergic agents.

Central dopaminergic transmission has been implicated in memory processes. The present experiments examined the effects of several direct acting dopaminergic agents on performance of a delayed-non-match-to-sample radial arm maze task. Preadministration of apomorphine (D1-D2 agonist; 0.25, 0.5, and 1.0 mg/kg), quinpirole (D2 agonist; 0.1 mg/kg), or SKF38393 (D1 agonist; 3 mg/kg) increased the latency of choices but did not affect any index of accuracy with a 1 h retention interval. Post-training administration of quinpirole (0.1, 0.2, 1.0, and 2.0 mg/kg), SKF38393 (0.3, 3.0, and 6.0 mg/kg), sulpiride (D2 antagonist; 3, 10, and 30 mg/kg), or SCH23390 (D1 antagonist; 0.01, 0.1, and 1.0 mg/kg) also did not affect accuracy, although quinpirole produced a dose-dependent increase in the latency of choices, assessed 10 h post-treatment. For comparison, pretraining and post-training administration of the benzodiazepine chlordiazepoxide (1, 3, 5 mg/kg) was also tested and produced dose-dependent impairments in mnemonic performance at either a 1 or 4 h retention interval. The effects of chlordiazepoxide are consistent with evidence indicating that GABAergic agents can influence memory processes. In contrast, the present findings indicate that (peripheral administration of dopaminergic agents IS) not sufficient to alter the mnemonic processes required for accurate performance of this DNMTS-RAM task.

Enhanced muscimol-induced behavioral responses after 6-OHDA lesions: relevance to susceptibility for self-mutilation behavior in neonatally lesioned rats.

Adult rats lesioned with 6-hydroxydopamine (6-OHDA), either as neonates or as adults, demonstrated increased turning, compared to unlesioned controls, when muscimol was unilaterally microinjected into the substantia nigra reticulata (SNR). At the higher doses of muscimol, the lesioned rats were so intensely lateralized that circling was impeded. These data suggest a functional supersensitivity of receptors associated with GABA function in the SNR of 6-OHDA-lesioned rats. When 30 ng muscimol was administered bilaterally into the SNR, self-mutilation behavior (SMB) was observed in 2/11 of the control unlesioned rats, in 0/8 adult 6-OHDA-lesioned rats, and in 11/11 of the neonatally-lesioned rats tested. The ability of muscimol to produce SMB in the rats lesioned as neonates was dose related. Behavioral observations indicated that behaviors associated with SMB (self-biting and taffy pulling) were present in neonatal, but not adult lesioned rats. Behavioral responses to dopamine agonist administration were also different between rats lesioned as neonates and those lesioned as adults with 6-OHDA. These data support the view that lesions of dopaminergic neurons cause an increased functional responsiveness of receptors acted upon by muscimol in the SNR, and that the increased susceptibility for SMB in neonatally lesioned rats is determined by neurons distal to the GABA receptor complex in the SNR.

Opioid modulation of ventral pallidal inputs.

While the ventral pallidum (VP) is known to be important in relaying information between the nucleus accumbens and target structures, it has become clear that substantial information processing occurs within the VP. We evaluated the possibility that opioid modulation of other transmitters contained in VP afferents is involved in this process. Initially, we demonstrated that opioids hyperpolarized VP neurons in vitro and suppressed spontaneous firing in vivo. The ability of opioids to modulate other transmitters was determined using microiontophoretically applied ligands and extracellular recordings of VP neurons from chloral hydrate-anesthetized rats. With neurons that responded to iontophoresed opioid agonists, the ejection current was reduced to a level that was below that necessary to alter spontaneous firing. This "subthreshold" current was used to determine the ability of mu opioid receptor (microR) agonists to alter VP responses to endogenous (released by electrical activation of afferents) and exogenous (iontophoretically applied) transmitters. microR agonists decreased the variability and enhanced the acuity (e.g., "signal-to-noise" relationship) of VP responses to activation of glutamatergic inputs from the prefrontal cortex and amygdala. By contrast, microR agonists attenuated both the slow excitatory responses to substance P and GABA-induced inhibitions that resulted from activating the nucleus accumbens. Subthreshold opioids also attenuated inhibitory responses to stimulating midbrain dopaminergic cells. These results suggest that a consequence of opioid transmission in the VP is to negate the influence of some afferents (e.g., midbrain dopamine and accumbal GABA and substance P) while selectively potentiating the efficacy of others (e.g., cortical and amygdaloid glutamate). Interpreted in the context of opiate abuse, microR opioids in the VP may serve to diminish the influence of reinforcement (ventral tegmental area and nucleus accumbens) in the transduction of cognition (prefrontal cortex) and affect (amygdala) into behavior. This may contribute to drug craving that occurs even in the absence of reward.

Effects of D1 and D2 antagonists on apomorphine-induced responses of ventral pallidal neurons.

The ventral pallidum with the adjacent substantia innominata (VP) has been described as a dopaminoceptive brain region. Repeated systemic injections of the dopamine agonist, apomorphine, induce dose-dependent alterations in VP neuronal activity. The present studies evaluated the contribution of D1 and D2 receptor subtypes to apomorphine-induced alterations in extracellularly recorded VP neuronal activity. Both sulpiride (D2 antagonist) and SCH23390 (D1 antagonist) attenuated many of these responses; however, pretreatment with either antagonist did not alter the number of responding neurons, or the maximal effect induced by apomorphine. Thus, activation of either receptor subtype by apomorphine is sufficient to initiate the observed responses, and both may be involved in dopaminergic modulation of VP neurons.

Effects of serotonergic 5-HT1A and 5-HT1B ligands on ventral pallidal neuronal activity.

To clarify the role of the 5-HT system in limbic outputs, the present study compared the effects of the 5-HT1A agonist 8-OH-DPAT and the 5-HT1B agonist CP-94253 with the non-selective 5-HT agonist TFMPP on the firing rate of ventral pallidal (VP) neurons recorded in chloral hydrate-anesthetized rats. 8-OH-DPAT (0.25-256 microg/kg i.v.) dose-dependently enhanced (9/26 neurons) or suppressed (8/26) activity, and the 5-HT1A antagonist (+)WAY-100135 often attenuated these responses. TFMPP (0.011-1.453 mg/kg i.v.) dose-dependently reduced the firing rate of 7/8 VP neurons tested. In contrast, CP-94253 (0.013-12.8 mg/kg i.v.) had little or no effect. In sum, these data suggest that the 5-HT1A receptor appears to be particularly important in influencing limbic outputs mediated via the VP.

Evaluations of ventral pallidal dopamine receptor activation in behaving rats.

Recent evidence justifies the inclusion of the ventral pallidum/substantia innominata (VP) into the category of dopaminoceptive brain regions. Since the VP is known to mediate both cognitive and motoric processes, the present study employed intracerebral microinjections of dopamine directly into the VP of rats to determine if the catecholamine influences these processes. Dopamine concentrations of up to 10 micrograms were ineffectual in altering performance parameters in a working memory task. However, concentrations as low as 0.01 micrograms increased locomotion in an open field. The magnitude of this response was related to the dopamine dose injected and the effect was attenuated by systemic pretreatment with the dopaminergic antagonist, flupentixol. These studies suggest that dopamine neurotransmission at the level of the VP may be important in the locomotor functions attributed to ascending dopamine systems.

Behavioral and biochemical assessment of time-related changes in globus pallidus and striatal dopamine induced by intranigrally administered neurotensin.

Microinjection of neurotensin (NT; 2 and 5 micrograms) into the substantia nigra zona compacta caused an increase in dopamine (DA) and DA metabolites in the rodent globus pallidus and striatum which persisted for at least 20 hours after peptide administration. Similar NT treatments given unilaterally into the nigra caused circling away from the injected side in amphetamine-pretreated rats, but were without effect when microinjected into saline-pretreated animals. Circling also occurred when the animals were given amphetamine 20 hours after intranigral NT administration. Contralateral rotation was observed with unilateral intranigral injections of gamma-hydroxybutyric acid (GHB; 400 micrograms) or with lower intranigral GHB doses (250 micrograms) in amphetamine-pretreated animals. The effects of GHB and NT differed in the manner in which the animals rotated as well as in the profile of DA and DA metabolite changes induced by these drugs. These studies indicated that: dopaminergic functions of the globus pallidus are influenced, like the striatum, by manipulations of the substantia nigra: NT and GHB likely act via different mechanisms to effect nigral dopamine-containing cells; and NT was capable of inducing changes in dopamine neurons which had long term consequences.