Age differences to methylphenidate-NAc neuronal and behavioral recordings from freely behaving animals.

Methylphenidate (MPD) is a psychostimulant that is widely prescribed to treat attention deficit-hyperactivity disorder, but it is abused recreationally as well. The nucleus accumbens (NAc) is part of the motivation circuit implicated in drug-seeking behaviors. The NAc neuronal activity was recorded alongside the behavioral activity from young and adult rats to determine if there are significant differences in the response to MPD. The same dose of MPD elicits behavioral sensitization in some animals and behavioral tolerance in others. In adult animals, higher doses of MPD resulted in a greater ratio of tolerance/sensitization. Animals who responded to chronic MPD with behavioral sensitization usually exhibited further increases in their NAc neuronal firing rates as well. Different upregulations of transcription factors (ΔFOSB/CREB), variable proportions of D1/D2 dopamine receptors, and modulation from other brain areas may predispose certain animals to express behavioral and neuronal sensitization versus tolerance to MPD.

Adult female rats' altered diurnal locomotor activity pattern following chronic methylphenidate treatment.

Methylphenidate (MPD) is one of the most prescribed pharmacological agents, which is also used for cognitive enhancement and recreational purposes. The objective of this study was to investigate the repetitive dose-response effects of MPD on circadian rhythm of locomotor activity pattern of female WKY rats. The hypothesis is that a change in the circadian activity pattern indicates a long-lasting effect of the drug. Four animal groups (saline control, 0.6, 2.5, and 10.0 mg/kg MPD dose groups) were housed in a sound-controlled room at 12:12 light/dark cycle. All received saline injections on experimental day 1 (ED 1). On EDs 2-7, the control group received saline injection; the other groups received 0.6, 2.5, or 10.0 mg/kg MPD, respectively. On ED 8-10, injections were withheld. On ED 11, each group received the same dose as EDs 2-7. Hourly histograms and cosine statistical analyses calculating the acrophase (ϕ), amplitude (A), and MESOR (M) were applied to assess the 24-h circadian activity pattern. The 0.6 and 2.5 mg/kg MPD groups exhibited significant (p < 0.05) change in their circadian activity pattern on ED 11. The 10.0 mg/kg MPD group exhibited tolerance on ED 11 and also a significant change in activity pattern on ED 8 compared to ED 1, consistent with withdrawal behavior (p < 0.007). In conclusion, chronic MPD administration alters circadian locomotor activity of adult female WKY rats and confirms that chronic MPD use elicits long-lasting effects.

Adolescent rats respond differently to methylphenidate as compared to adult rats- concomitant VTA neuronal and behavioral Recordings.

Methylphenidate (MPD) is the most widely prescribed psychostimulant used in adolescents and adults to treat attention-deficit/hyperactivity disorder (ADHD). The recreational use of MPD is becoming more prevalent because of its ability to improve cognitive enhancement. The ventral tegmental area (VTA) of the brain is highly associated with reward, cognition and addiction to drugs including psychostimulants like MPD. The VTA neuronal activity was recorded alongside the horizontal behavioral activity from freely behaving non-anesthetized rats. Four adolescent and four adult groups were treated with either saline, 0.6, 2.5 or 10.0 mg/kg MPD. In both adolescent and adult animals, the animals responded to MPD in a dose-dependent manner, such that as the dose of MPD increased, more animals and more VTA unit responded to the drug. The same doses of MPD elicited in some animals behavioral and neuronal sensitization and in other animals behavioral and neuronal tolerance. In the 0.6 and 10.0 mg/kg MPD dose groups there were significant differences between the age groups for how many animals expressed behavioral sensitization and behavioral tolerance to chronic MPD exposure. Additionally, the animal's behavioral response to MPD by excitation or attenuation of activity did not always correlate to the VTA neuronal response, and the age group with significantly higher behavioral responses did not always correlate to the age group with significantly higher VTA neuronal responses for a given MPD dose. These findings differ from similar studies recorded from the prefrontal cortex (PFC), which exhibited behavioral responses continuously directly correlated to PFC responses for increasing MPD doses. This demonstrates that unlike other areas of the brain, there is not a direct relationship between VTA firing and behavioral activity, suggesting that there is input or modulation of this area from elsewhere in the brain. Further investigation is needed to clearly understand the relationship between VTA firing rates and behavioral responses to different MPD doses, especially given the significant differences in response between young and adult animals and the increasing use of the drug in adolescent populations.

Methylphenidate and amphetamine modulate differently the NMDA and AMPA glutamatergic transmission of dopaminergic neurons in the ventral tegmental area.

Behavioral and neurochemical studies suggest that the induction of behavioral sensitization to psychostimulants involves transient changes at the synapses of the ventral tegmental area's dopaminergic neurons (VTA-DA). Differences in the behavioral response to amphetamine (Amph) and methylphenidate (MPD) were observed. In an attempt to understand these behavioral differences at the neuronal level, the dose-response characteristics of these two psychostimulants on electrophysiologically identified VTA-DA neurons at the glutamatergic synapse were investigated. Miniature excitatory post-synaptic currents (mEPSCs) and electrically induced EPSCs were recorded from horizontal midbrain slices of rats that had been pretreated intraperitoneally (i.p.) with saline (control), Amph (2.5, 5.0, 10.0 or 20.0 mg/kg), or MPD (2.5, 5.0, 10.0 or 20.0 mg/kg) 24 h before the recording. Perfusion of Amph through the bath (2.5, 5.0, 10.0 or 20.0 microM) increased the frequency (p<0.01) and the amplitude (p<0.05) of mEPSCs in dose-response characteristics, while MPD perfusion through the bath (2.5, 5.0, 10.0, or 20.0 microM) increased only the frequency (p<0.05) of the mEPSC. Both psychostimulants increased the prefrontal cortex's (PFC) glutamatergic EPSC in the VTA-DA neurons. However, only the higher doses of MPD induced significant effects (p<0.05) on the N-methyl-D-aspartate (NMDA) receptor-mediated EPSC but had no effects on the EPSC mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA/kainate) receptors. Contrarily, Amph increased both kinds of mediated EPSC, but mainly the EPSC mediated by AMPA/kainate receptors (p<0.01). These electrophysiological differences could represent the underlying mechanism responsible for the differences of behavioral effects, such as behavioral sensitization, elicited by MPD and Amph.

Dorsal raphe stimulation reduces responses of parafascicular neurons to noxious stimulation.

Spontaneous activity, responses to noxious stimulation and response to electrical stimulation of the dorsal raphe were recorded extracellularly from single units in the parafascicularis (PF) nucleus in the rat. Three types of spontaneous activity were found: 'slow' firing units (1-10 spikes/sec), 'bursting' units (bursts of 2-5 spikes/10-20 msec, bursts repeat every 1-2 sec), and 'fast' firing units (15-40 spikes/sec). Noxious stimulation increased the firing rate of 63% of the slow cells and 87% of the bursting cells, while the fast firing units did not respond. Dorsal raphe (DR) stimulation decreased the firing rates of both the slow and bursting PF cells only. The degree of suppression of PF units was directly related to the frequency and intensity of the DR stimulation. When the noxious stimulus was combined with DR stimulation, DR stimulation inhibited the increase in firing rate caused by the noxious stimulus. The firing rate during combined DR stimulation and noxious stimulation averaged 51% of that during the noxious stimulation alone. In several units, DR stimulation had no effect on spontaneous activity when applied alone but did decrease the effects of noxious stimulation when applied simultaneously. The results indicate that in addition to other possible mechanisms, the DR may affect responses to noxious stimuli via an ascending modulation pathway to the parafascicularis nucleus in the thalamus.

Interferon modifies morphine withdrawal phenomena in rodents.

Interferon injection in morphine dependent rats prior to naloxone treatment eliminates 7 behavioral sings of the abstinence (withdrawal) phenomenon. When a single injection of interferon was given prior to chronic morphine treatment it reduces opiate addiction liability.

Responses of neurons in bed nucleus of the stria terminalis to microiontophoretically applied morphine, norepinephrine and acetylcholine.

The bed nucleus of the stria terminalis is a basal forebrain nucleus that receives inputs from limbic system nuclei and sends projections to several hypothalamic and brainstem nuclei, proposed to be involved in the physiological response to stressors. The bed nucleus of the stria terminalis also receives norepinephrine- (NE), acetylcholine- (ACh) and opiate-containing projections. The objectives of this experiment were to examine the effects of microiontophoretically applied ACh, NE and morphine sulfate (M) on neurons of the bed nucleus in urethane-anesthetized rats and to determine the degree to which these systems interact on individual neurons. Acetylcholine was excitatory in 43% of the neurons and NE was inhibitory in 70%. Morphine however, elicited two distinct types of response--excitation in 24% of the neurons and inhibition in 28%. Each of these effects was current-dependent and monophasic. Since 77% of neurons of the bed nucleus responded to two or more of the drugs used in this experiment, statistical analysis was used to determine correlations between responses to each of these drugs. This analysis showed that neurons that responded to M with excitation also responded to ACh with excitation, while neurons that responded to M with inhibition, were nonresponsive to ACh. No correlation was found between NE and either M or ACh. Thus, this experiment demonstrated the possible effects of noradrenergic, cholinergic and opioid innervation on firing rates of neurons of the bed nucleus of the stria terminalis and may explain the roles these neurotransmitters play in modulating the response to stress.

Evidence that opiate addiction is in part an immune response. Destruction of the immune system by irradiation-altered opiate withdrawal.

Experiments were performed to determine whether physical dependence on opiates (CNS phenomena) can be altered by destruction of the immune system. Irradiation, prior to or after chronic treatment with morphine significantly reduced the opiate-withdrawal syndrome as assessed by naloxone-induced abstinence. This study supports the proposition that addiction to opiates is related, at least in part, to interaction between the central nervous system (CNS) and the immune system.

Microiontophoretic application of muramyl-dipeptide upon single cortical, hippocampal and hypothalamic neurons in rats.

Muramyl-dipeptide (MDP), a metabolite of bacterial cell walls, has a variety of biological effects, including the induction of acute phase serum glycoproteins and fever, and the promotion of slow wave sleep. Muramyl-dipeptide and other products, derived from immune responses, may act directly in the CNS to recruit secondary autonomic and endocrine responses to disease. To test this hypothesis the properties of single neuron discharges, following local application (microiontophoresis) of MDP within the somatosensory cortex, the dorsal hippocampus and the medial basal hypothalamus were studied in rats. The results obtained from cortical (N = 30), hippocampal (N = 28) and hypothalamic (N = 32) neurons demonstrated a direct effect of MDP upon all three regions of the brain. In addition, MDP modified the responses of these same neurons to morphine. These results support a role of MDP in the process of neuro-immune modulation and further demonstrate an interaction between lymphoid agents and opioids in the CNS.

Response characteristics of thalamic neurons to microiontophoretically applied morphine.

The extracellular unit activity of parafasciculus thalamic neurons was studied before and after incremental microiontophoretic applications of morphine and naloxone. The parafasciculus neurons could be divided into three groups according to their initial spontaneous discharges. Only two groups responded to morphine as a function of dose and, within these groups, there were 12 different patterns. These findings suggest that the parafasciculus neurons responded in a heterogenous manner to morphine and they also provide physiological support for the biochemical evidence indicating the existence of multiple opiate receptors.

Alpha-interferon suppresses food intake and neuronal activity of the lateral hypothalamus.

Alpha-interferon (alpha-IFN) treatment in humans induces anorexic effects. However, the mechanisms and sites of action are unknown. Rats implanted with an intracerebroventricular (i.c.v.) cannula for local injection, and semi-microelectrodes in the lateral hypothalamic (LH) area for neuronal recording were used. The animals were kept in metabolic cages, and food and water intake was measured daily at 7:00 and 19:00 hr for 35 days, including: 5 days before the experiment; 10 days during daily alpha-IFN application (either i.p. 1500 I.U./gbw, or i.c.v. 1500 and 150 I.U./animal) and/or a vehicle control group; and 20 days post drug treatment. The unitary activity recording from the LH area was made before (30 min), during (10 min) and after (200 min) the alpha-IFN applications. alpha-IFN elicited a reversible dose-related decrease of both food intake and body weight. This decrease in food intake following alpha-IFN injections was correlated with a depression of LH neuronal electrical activity. Since direct brain application (i.c.v.) and systemic (i.p.) alpha-IFN treatment elicited identical responses, it is possible to assume that alpha-IFN suppresses food intake by a direct action on CNS sites including the LH neurons.

Blockade of sensitization to methylphenidate by MK-801: partial dissociation from motor effects.

The role of MK-801's locomotor effect in blocking the development of sensitization to methylphenidate was investigated utilizing a computerized locomotor activity monitoring system. After 7 days of acclimation to a 12:12 light-dark cycle (lights on at 07:00), male Sprague-Dawley rats (n=62) were housed in test cages and motor activity was recorded continuously for 16 days. The first 2 days of recording served as a baseline for each rat, and on day 3 each rat received a saline injection. On days 4 to 9 rats were randomly divided into seven groups: Rats received either six daily s.c. injections of methylphenidate (2.5 mg/kg; Group 1), or six daily i.p. injections of 0.30 mg/kg, 0.05 mg/kg MK-801 (Groups 2 and 3, respectively); two MK-801 pre-treatment groups received a single i.p. injection of 0.05, or 0.30 mg/kg MK-801 one hour prior to 2.5 mg/kg methylphenidate (n=8 each) on day 4 followed by five daily injections of 2.5 mg/kg methylphenidate; and finally, two cotreatment groups received a challenge dose of 2.5 mg/kg methylphenidate on day 4 followed by either 0.05 or 0.30 mg/kg MK-801 i.p. one hour prior to 2.5 mg/kg methylphenidate from days 5 to 9. All groups were allowed five days of no treatment before being re-challenged on day 15 with the same treatment they received on day 4. Methylphenidate and 0.30 mg/kg MK-801 sensitized to their own locomotor effects, but 0.05 mg/kg MK-801, which had no acute motor effects, did not. The administration of MK-801 (0.30 mg/kg) prior to methylphenidate either singly on day 4, or coadministered throughout the repeated methylphenidate treatment phase, blocked the development of sensitization to methylphenidate. However, MK-801 at 0.05 mg/kg delayed the development of sensitization when co-administered on days 5 to 9, but a single injection 1 h prior to methylphenidate on day 4 did not prevent sensitization to subsequent methylphenidate administration. In conclusion, MK-801 prevents sensitization to methylphenidate; motor stimulation by MK-801 is not necessary for short-term prevention or delay of sensitization to methylphenidate but may be necessary for a persistent blockade of sensitization.

Microiontophoretic application of morphine and naloxone to neurons in hypothalamus of rat.

The present experiments used urethane-anesthetized rats and single cell recording to study the electrophysiological properties of ventromedial hypothalamic (VMH) cells following different doses of morphine and naloxone, applied microiontophoretically. More than 45% of ventromedial hypothalamic units reacted in a dose-response fashion to local application of morphine. In the majority of the ventromedial hypothalamic neurons, naloxone failed to reverse the effects of morphine. Naloxone alone had effects on 37% of the ventromedial hypothalamic units. The ventromedial hypothalamic units exhibited different response patterns from those observed from other CNS sites in response to the microiontophoretic application of morphine and naloxone; this difference is discussed. The present neurophysiological findings support the existence of opiate target sites with multiple opiate receptors within the ventromedial hypothalamus.

Prolonged daily inhalation of halothane modifies the dose-response pattern to acute administration of halothane. An electrophysiological study.

Sensory-evoked field potentials were obtained from freely moving rats implanted sterotaxically with permanent electrodes in the parafasciculus thalami (PF), mesencephalic central gray (CG), ventromedial hypothalamus (VMH) and somatosensory cortex (SCX). Animals were exposed to chronic, subanesthetic inhalation of halothane (0.5%, 3 hr/day, 5 days/week) for 56 days. The averaged acoustic evoked responses (AAER) were recorded on day 0, as well as at 28 and 56 days after a 48-hr halothane-free period ("control") and after acute doses of halothane (0.25, 0.5 and 1.5%). In general, the averaged sensory-evoked responses from each structure were affected at day 0 of the experiment in dose-response manner, and suppression of the responses was the main effect of halothane. Chronic exposure to subanesthetic inhalation of halothane produced marked alteration of the "control" recording from 3 CNS structures; mainly from the mesencephalic central gray, the parafasciculus thalami and the somatosensory cortex and the direction (increase or decrease) of the averaged acoustic evoked responses in all the four CNS sites studied. The total responsiveness was modified as well, i.e. the recordings obtained from the mesencephalic central gray and somatosensory cortex exhibited hypersensitivity while the recordings obtained from the parafasciculus thalami and ventromedial hypothalamus exhibited tolerance. It is concluded that prolonged and intermittent inhalation of halothane can alter the electrophysiological properties of the four structures investigated.

Effects of microiontophoretic application of cocaine, alone and with receptor antagonists, upon the neurons of the medial prefrontal cortex, nucleus accumbens and caudate nucleus of rats.

The spontaneous extracellular electrical activity of 102 neurons, within the caudate nucleus (CN), medial prefrontal cortex (MPC), nucleus accumbens (NAc) and a control site, the lateral thalamic nucleus (LT), was studied. Cocaine depressed spontaneous activity in the majority of the cells studied from all regions except the lateral thalamus. Desipramine, which has been used clinically for the treatment of withdrawal of cocaine, also depressed neuronal activity in the caudate nucleus. In addition, of the three receptor antagonists tested, sulpiride, methysergide and naloxone, only the dopamine antagonist (sulpiride) affected cocaine-induced neuronal responses. This study further emphasizes the emerging importance of midbrain dopaminergic systems in the pharmacological effects of this important drug of abuse.

Differential modification of morphine and methadone dependence by interferon alpha.

Subcutaneous implantation of a pellet of methadone was presented as a novel method for the establishment of physical dependence upon this agent and it was compared to (1) the state of physical dependence induced by multiple injections of methadone, administered over several days, and (2) the dependence established by injections of morphine and the implantation of a morphine pellet. Comparable signs of drug dependence were observed in rats treated with both morphine and methadone following the administration of the opiate antagonist naloxone. The administration of interferon-alpha significantly attenuated the severity of the withdrawal syndrome in dependent rats after chronic exposure to morphine and to a lesser extent after morphine and methadone in combination. In contrast, alpha interferon did not affect 6 of the 7 abstinence signs in animals dependent upon methadone alone. The observations suggest that the states of physical dependence upon morphine and methadone may be separate phenomena that involve different physiological mechanisms. Thus, interferon may be a useful adjunct in the treatment of subjects dependent upon morphine but not in those dependent on methadone.

Muramyl-dipeptide, a macrophage-derived cytokine, alters neuronal activity in hypothalamus and hippocampus but not in the dorsal raphe/periaqueductal gray of rats.

Muramyl-dipeptide (MDP) is derived in vivo by degradation of bacteria cell walls and is the minimum fragment that stimulates the acute phase response to bacterial infection. The present study investigates whether this specific product of an immune response affects central nervous system (CNS) function. To this end, the activity of single neurons within the hypothalamus, hippocampus, and dorsal raphe/periaqueductal gray region prior to and following systemic (i.p.) injection was studied. The results obtained from a total of 120 cells demonstrate that single hypothalamic and hippocampal neurons, sites previously shown to aid in the integration of various environmental stimuli into physiologic processes, alter their neuronal activity in site-specific manners following MDP administration. The specificity of the responses included both the threshold for activation of particular sites, effects of increasing dosages upon response pattern characteristics, and time course to the changes observed. These results therefore suggest that MDPs may play a role in the neuro-immunologic regulatory pathways during the immune response to bacterial infection.

Does the immune system communicate with the central nervous system? Interferon modifies central nervous activity.

The present investigation determined whether an immunomodulator agent modified the central nervous system activity as measured behaviorally and neurophysiologically. Two types of interferons (IFNs), alpha (alpha) and gamma (gamma), were applied locally (microiontophoretically) into various regions of the rat brain simultaneously with single neuron recording from the cerebral cortex, hippocampus, thalamus and hypothalamus. Of the various IFNs, only alpha-IFN altered single cell activity in all brain structures in a dose-dependent manner. Moreover, systemic administration of alpha-IFN altered the naloxone-induced abstinence syndrome in morphine-dependent rats. These observations suggest that immunomodulators such as alpha-IFN are capable of influencing directly central nervous system function as well as the immune system.

Evidence of neuro-immunologic interactions: cyclosporine modifies opiate withdrawal by effects on the brain and immune components.

The capacity of the immune system to participate in processes primarily considered to be central nervous system (CNS) phenomena has been suggested recently by several studies demonstrating the ability of various immune modifiers to attenuate opiate withdrawal severity. The present study demonstrates that within 2 h after injection, the immune modifier cyclosporine A (CsA) has the ability to attenuate the opiate withdrawal syndrome precipitated by naloxone in morphine-dependent animals. Furthermore, it is demonstrated that this effect of CsA can be adoptively transferred by splenic mononuclear cells from immune-modulated (CsA-treated) donors into morphine-dependent recipients. However, unlike direct injections of CsA, CsA-treated immune components require at least 24 h to achieve their full attenuating effect upon withdrawal severity. Since opiate withdrawal behavior is predominantly a CNS-mediated phenomenon, these observations suggest both direct effects of CsA on the brain as well as the participation of immune components in the opiate withdrawal syndrome. This finding lends further support to the hypothesis that immune components have the ability to modulate central nervous system activities in a neuro-immunologic axis of communication.

The brain and the immune system: an intact immune system is essential for the manifestation of withdrawal in opiate addicted rats.

Gamma irradiation (500 rad) is often used to suppress the immune system in mice, rats and man. Recently, it was shown that irradiation prior to chronic morphine treatment, dramatically reduces the severity of naloxone-precipitated withdrawal in morphine-dependent animals. In the present study adoptive transfer of 2-6 x 10(8) splenocytes to irradiated rats prior to chronic morphine treatment restored the severity of all withdrawal signs precipitated by naloxone. In contrast, adoptive transfer of fractionated splenocyte subpopulations only partially restored withdrawal severity; and transfer of irradiated splenocytes, red blood cells or diluted numbers of normal splenocytes did not have any observed restorative effect. These findings suggest that specific cellular activities or factors derived from lymphoid cells are required for the expression of opiate withdrawal.