Flow Cytometry of Synaptoneurosomes (FCS) Reveals Increased Ribosomal S6 and Calcineurin Proteins in Activated Medial Prefrontal Cortex to Nucleus Accumbens Synapses.

Learning and behavior activate cue-specific patterns of sparsely distributed cells and synapses called ensembles that undergo memory-encoding engram alterations. While Fos is often used to label selectively activated cell bodies and identify neuronal ensembles, there is no comparable endogenous marker to label activated synapses and identify synaptic ensembles. For the purpose of identifying candidate synaptic activity markers, we optimized a flow cytometry of synaptoneurosome (FCS) procedure for assessing protein alterations in activated synapses from male and female rats. After injecting yellow fluorescent protein (YFP)-expressing adeno-associated virus into medial prefrontal cortex (mPFC) to label terminals in nucleus accumbens (NAc) of rats, we injected 20 mg/kg cocaine in a novel context (cocaine+novelty) to activate synapses, and prepared NAc synaptoneurosomes 0-60 min following injections. For FCS, we used commercially available antibodies to label presynaptic and postsynaptic markers synaptophysin and PSD-95 as well as candidate markers of synaptic activity [activity-regulated cytoskeleton protein (Arc), CaMKII and phospho-CaMKII, ribosomal protein S6 (S6) and phospho-S6, and calcineurin and phospho-calcineurin] in YFP-labeled synaptoneurosomes. Cocaine+novelty increased the percentage of S6-positive synaptoneurosomes at 5-60 min and calcineurin-positive synaptoneurosomes at 5-10 min. Electron microscopy verified that S6 and calcineurin levels in synaptoneurosomes were increased 10 min after cocaine+novelty. Pretreatment with the anesthetic chloral hydrate blocked cocaine+novelty-induced S6 and calcineurin increases in synaptoneurosomes, and novel context exposure alone (without cocaine) increased S6, both of which indicate that these increases were due to neural activity per se. Overall, FCS can be used to study protein alterations in activated synapses coming from specifically labeled mPFC projections to NAc.SIGNIFICANCE STATEMENT Memories are formed during learning and are stored in the brain by long-lasting molecular and cellular alterations called engrams formed within specific patterns of cue-activated neurons called neuronal ensembles. While Fos has been used to identify activated ensemble neurons and the engrams within them, we have not had a similar marker for activated synapses that can be used to identify synaptic engrams. Here we developed a procedure for high-throughput in-line analysis of flow cytometry of synaptoneurosome (FCS) and found that ribosomal S6 protein and calcineurin were increased in activated mPFC-NAc synapses. FCS can be used to study protein alterations in activated synapses within specifically labeled circuits.

Effect of Selective Lesions of Nucleus Accumbens µ-Opioid Receptor-Expressing Cells on Heroin Self-Administration in Male and Female Rats: A Study with Novel Oprm1-Cre Knock-in Rats.

The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to MOR-expressing cells. After performing anatomic and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to study the involvement of NAc MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and FISH chain reaction (HCR-FISH), we found no differences in Oprm1 expression in NAc, dorsal striatum, and dorsal hippocampus, or MOR receptor density (except dorsal striatum) or function between Oprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed that iCre is highly coexpressed with Oprm1 (95%-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells. We found that the lesions decreased acquisition of heroin self-administration in male Oprm1-Cre rats and had a stronger inhibitory effect on the effort to self-administer heroin in female Oprm1-Cre rats. The validation of an Oprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study indicates that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in male and female rats.SIGNIFICANCE STATEMENT The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to brain MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to show that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in males and females. The new Oprm1-Cre rats can be used to study the role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.

SMART: An Open-Source Extension of WholeBrain for Intact Mouse Brain Registration and Segmentation.

Mapping immediate early gene (IEG) expression across intact mouse brains allows for unbiased identification of brain-wide activity patterns underlying complex behaviors. Accurate registration of sample brains to a common anatomic reference is critical for precise assignment of IEG-positive ("active") neurons to known brain regions of interest (ROIs). While existing automated voxel-based registration methods provide a high-throughput solution, they require substantial computing power, can be difficult to implement and fail when brains are damaged or only partially imaged. Additionally, it is challenging to cross-validate these approaches or compare them to any preexisting literature based on serial coronal sectioning. Here, we present the open-source R package SMART (Semi-Manual Alignment to Reference Templates) that extends the WholeBrain R package framework to automated segmentation and semi-automated registration of intact mouse brain light-sheet fluorescence microscopy (LSFM) datasets. The SMART package was created for novice programmers and introduces a streamlined pipeline for aligning, registering, and segmenting LSFM volumetric datasets across the anterior-posterior (AP) axis, using a simple "choice game" and interactive menus. SMART provides the flexibility to register whole brains, partial brains or discrete user-chosen images, and is fully compatible with traditional sectioned coronal slice-based analyses. We demonstrate SMART's core functions using example datasets and provide step-by-step video tutorials for installation and implementation of the package. We also present a modified iDISCO+ tissue clearing procedure for uniform immunohistochemical labeling of the activity marker Fos across intact mouse brains. The SMART pipeline, in conjunction with the modified iDISCO+ Fos procedure, is ideally suited for examination and orthogonal cross-validation of brain-wide neuronal activation datasets.

Morphine and Fentanyl Repeated Administration Induces Different Levels of NLRP3-Dependent Pyroptosis in the Dorsal Raphe Nucleus of Male Rats via Cell-Specific Activation of TLR4 and Opioid Receptors.

Morphine promotes neuroinflammation after NOD-like receptor protein 3 (NLRP3) oligomerization in glial cells, but the capacity of other opioids to induce neuroinflammation and its relationship to the development of analgesic tolerance is unknown. We studied the effects of morphine and fentanyl on NLRP3 inflammasome activation in glial and neuronal cells in the dorsal raphe nucleus (DRN), a region involved in pain regulation. Male Wistar rats received i.p. injections of morphine (10 mg/kg) or fentanyl (0.1 mg/kg) 3 × daily for 7 days and were tested for nociception. Two hours after the last (19th) administration, we analyzed NLRP3 oligomerization, caspase-1 activation and gasdermin D-N (GSDMD-N) expression in microglia (CD11b positive cells), astrocytes (GFAP-positive cells) and neurons (NeuN-positive cells). Tolerance developed to both opioids, but only fentanyl produced hyperalgesia. Morphine and fentanyl activated NLRP3 inflammasome in astrocytes and serotonergic (TPH-2-positive) neurons, but fentanyl effects were more pronounced. Both opioids increased GFAP and CD11b immunoreactivity, caspase-1 and GSDMD activation, indicating pyroptotic cell death. The opioid receptor antagonist (-)-naloxone, but not the TLR4 receptor antagonist (+)-naloxone, prevented microglia activation and NLRP3 oligomerization. Only (+)-naloxone prevented astrocytes' activation. The anti-inflammatory agent minocycline and the NLRP3 inhibitor MCC950 delayed tolerance to morphine and fentanyl antinociception and prevented fentanyl-induced hyperalgesia. MCC950 also prevented opioid-induced NLRP3 oligomerization. In conclusion, morphine and fentanyl differentially induce cell-specific activation of NLRP3 inflammasome and pyroptosis in the DRN through TLR4 receptors in astrocytes and through opioid receptors in neurons, indicating that neuroinflammation is involved in opioid-induced analgesia and fentanyl-induced hyperalgesia after repeated administrations.

Relapse-Associated Transient Synaptic Potentiation Requires Integrin-Mediated Activation of Focal Adhesion Kinase and Cofilin in D1-Expressing Neurons.

Relapse to drug use can be initiated by drug-associated cues. The intensity of cue-induced drug seeking in rodent models correlates with the induction of transient synaptic potentiation (t-SP) at glutamatergic synapses in the nucleus accumbens core (NAcore). Matrix metalloproteinases (MMPs) are inducible endopeptidases that degrade extracellular matrix (ECM) proteins, and reveal tripeptide Arginine-Glycine-Aspartate (RGD) domains that bind and signal through integrins. Integrins are heterodimeric receptors composed of αß subunits, and a primary signaling kinase is focal adhesion kinase (FAK). We previously showed that MMP activation is necessary for and potentiates cued reinstatement of cocaine seeking, and MMP-induced catalysis stimulates ß3-integrins to induce t-SP. Here, we determined whether ß3-integrin signaling through FAK and cofilin (actin depolymerization factor) is necessary to promote synaptic growth during t-SP. Using a small molecule inhibitor to prevent FAK activation, we blocked cued-induced cocaine reinstatement and increased spine head diameter (dh). Immunohistochemistry on NAcore labeled spines with ChR2-EYFP virus, showed increased immunoreactivity of phosphorylation of FAK (p-FAK) and p-cofilin in dendrites of reinstated animals compared with extinguished and yoked saline, and the p-FAK and cofilin depended on ß3-integrin signaling. Next, male and female transgenic rats were used to selectively label D1 or D2 neurons with ChR2-mCherry. We found that p-FAK was increased during drug seeking in both D1 and D2-medium spiny neurons (MSNs), but increased p-cofilin was observed only in D1-MSNs. These data indicate that ß3-integrin, FAK and cofilin constitute a signaling pathway downstream of MMP activation that is involved in promoting the transient synaptic enlargement in D1-MSNs induced during reinstated cocaine by drug-paired cues.SIGNIFICANCE STATEMENT Drug-associated cues precipitate relapse, which is correlated with transient synaptic enlargement in the accumbens core. We showed that cocaine cue-induced synaptic enlargement depends on matrix metalloprotease signaling in the extracellular matrix (ECM) through ß3-integrin to activate focal adhesion kinase (FAK) and phosphorylate the actin binding protein cofilin. The nucleus accumbens core (NAcore) contains two predominate neuronal subtypes selectively expressing either D1-dopamine or D2-dopamine receptors. We used transgenic rats to study each cell type and found that cue-induced signaling through cofilin phosphorylation occurred only in D1-expressing neurons. Thus, cocaine-paired cues initiate cocaine reinstatement and synaptic enlargement through a signaling cascade selectively in D1-expressing neurons requiring ECM stimulation of ß3-integrin-mediated phosphorylation of FAK (p-FAK) and cofilin.

Focal transcranial magnetic stimulation in awake rats: Enhanced glucose uptake in deep cortical layers.

BACKGROUND: Transcranial magnetic stimulation (TMS) is an emerging neuromodulation tool. However, preclinical models of TMS are limited. OBJECTIVE: To develop a method for performing TMS in awake rats and to characterize neuronal response to TMS by mapping glucose uptake following TMS administration. METHODS: A headpost was implanted into rat skull serving as a refence to guide TMS target. Motor threshold measurement was used as the metric to assess the consistency in TMS delivery across animals and across sessions. Using a fluorescent glucose analogue (2-NBDG) as a marker of neuronal activity, we mapped glucose uptake in response to TMS of the rat motor cortex. RESULTS: The average motor threshold (n = 41) was 34.6 ± 6.3 % of maximum stimulator output (MSO). The variability of motor threshold across animals was similar to what has been reported in human studies. Furthermore, there was no significant difference in motor threshold measured across 3 separate days. Enhancement in fluorescent signals were TMS dose (power)-dependent, which centered around the motor cortex, covering an area medial-laterally 2 mm, rostral-caudally 4 mm at 55 % MSO, and 3 mm at 35 % MSO. The count of total cells with significant fluorescent signal was: 107 ± 23 (55 % MSO), 73 ± 11 (35 % MSO) and 42 ± 11 (sham, 5% MSO). CONCLUSIONS: Our method allows for consistent motor threshold assessment for longitudinal studies. Notably, cells with fluorescent signal enhancement were consistently aggregated in deep cortical layers, with minimal enhancement in superficial layers COMPARISONS WITH EXISTING METHOD(S): To our knowledge, this is the first study of focal TMS in awake rodents.

Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.

Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.

Dorsal Raphe Dual Serotonin-Glutamate Neurons Drive Reward by Establishing Excitatory Synapses on VTA Mesoaccumbens Dopamine Neurons.

Dorsal raphe (DR) serotonin neurons provide a major input to the ventral tegmental area (VTA). Here, we show that DR serotonin transporter (SERT) neurons establish both asymmetric and symmetric synapses on VTA dopamine neurons, but most of these synapses are asymmetric. Moreover, the DR-SERT terminals making asymmetric synapses on VTA dopamine neurons coexpress vesicular glutamate transporter 3 (VGluT3; transporter for accumulation of glutamate for its synaptic release), suggesting the excitatory nature of these synapses. VTA photoactivation of DR-SERT fibers promotes conditioned place preference, elicits excitatory currents on mesoaccumbens dopamine neurons, increases their firing, and evokes dopamine release in nucleus accumbens. These effects are blocked by VTA inactivation of glutamate and serotonin receptors, supporting the idea of glutamate release in VTA from dual DR SERT-VGluT3 inputs. Our findings suggest a path-specific input from DR serotonergic neurons to VTA that promotes reward by the release of glutamate and activation of mesoaccumbens dopamine neurons.

Lateral Hypothalamic GABAergic Neurons Encode Reward Predictions that Are Relayed to the Ventral Tegmental Area to Regulate Learning.

Eating is a learned process. Our desires for specific foods arise through experience. Both electrical stimulation and optogenetic studies have shown that increased activity in the lateral hypothalamus (LH) promotes feeding. Current dogma is that these effects reflect a role for LH neurons in the control of the core motivation to feed, and their activity comes under control of forebrain regions to elicit learned food-motivated behaviors. However, these effects could also reflect the storage of associative information about the cues leading to food in LH itself. Here, we present data from several studies that are consistent with a role for LH in learning. In the first experiment, we use a novel GAD-Cre rat to show that optogenetic inhibition of LH γ-aminobutyric acid (GABA) neurons restricted to cue presentation disrupts the rats' ability to learn that a cue predicts food without affecting subsequent food consumption. In the second experiment, we show that this manipulation also disrupts the ability of a cue to promote food seeking after learning. Finally, we show that inhibition of the terminals of the LH GABA neurons in ventral-tegmental area (VTA) facilitates learning about reward-paired cues. These results suggest that the LH GABA neurons are critical for storing and later disseminating information about reward-predictive cues.

Specificity and impact of adrenergic projections to the midbrain dopamine system.

Dopamine (DA) is a neuromodulator that regulates different brain circuits involved in cognitive functions, motor coordination, and emotions. Dysregulation of DA is associated with many neurological and psychiatric disorders such as Parkinson's disease and substance abuse. Several lines of research have shown that the midbrain DA system is regulated by the central adrenergic system. This review focuses on adrenergic interactions with midbrain DA neurons. It discusses the current neuroanatomy including source of adrenergic innervation, type of synapses, and adrenoceptors expression. It also discusses adrenergic regulation of DA cell activity and neurotransmitter release. Finally, it reviews several neurological and psychiatric disorders where changes in adrenergic system are associated with dysregulation of the midbrain DA system. This article is part of a Special Issue entitled SI: Noradrenergic System.

Prefrontal Cortical Kappa Opioid Receptors Attenuate Responses to Amygdala Inputs.

Kappa opioid receptors (KORs) have been implicated in anxiety and stress, conditions that involve activation of projections from the basolateral amygdala (BLA) to the medial prefrontal cortex (mPFC). Although KORs have been studied in several brain regions, their role on mPFC physiology and on BLA projections to the mPFC remains unclear. Here, we explored whether KORs modify synaptic inputs from the BLA to the mPFC using in vivo electrophysiological recordings with electrical and optogenetic stimulation. Systemic administration of the KOR agonist U69,593 inhibited BLA-evoked synaptic responses in the mPFC without altering hippocampus-evoked responses. Intra-mPFC U69,593 inhibited electrical and optogenetic BLA-evoked synaptic responses, an effect blocked by the KOR antagonist nor-BNI. Bilateral intra-mPFC injection of the KOR antagonist nor-BNI increased center time in the open field test, suggesting an anxiolytic effect. The data demonstrate that mPFC KORs negatively regulate glutamatergic synaptic transmission in the BLA-mPFC pathway and anxiety-like behavior. These findings provide a framework whereby KOR signaling during stress and anxiety can regulate the flow of emotional state information from the BLA to the mPFC.

A subpopulation of neurochemically-identified ventral tegmental area dopamine neurons is excited by intravenous cocaine.

Systemic administration of cocaine is thought to decrease the firing rates of ventral tegmental area (VTA) dopamine (DA) neurons. However, this view is based on categorizations of recorded neurons as DA neurons using preselected electrophysiological characteristics lacking neurochemical confirmation. Without applying cellular preselection, we recorded the impulse activity of VTA neurons in response to cocaine administration in anesthetized adult rats. The phenotype of recorded neurons was determined by their juxtacellular labeling and immunohistochemical detection of tyrosine hydroxylase (TH), a DA marker. We found that intravenous cocaine altered firing rates in the majority of recorded VTA neurons. Within the cocaine-responsive neurons, half of the population was excited and the other half was inhibited. Both populations had similar discharge rates and firing regularities, and most neurons did not exhibit changes in burst firing. Inhibited neurons were more abundant in the posterior VTA, whereas excited neurons were distributed evenly throughout the VTA. Cocaine-excited neurons were more likely to be excited by footshock. Within the subpopulation of TH-positive neurons, 36% were excited by cocaine and 64% were inhibited. Within the subpopulation of TH-negative neurons, 44% were excited and 28% were inhibited. Contrary to the prevailing view that all DA neurons are inhibited by cocaine, we found a subset of confirmed VTA DA neurons that is excited by systemic administration of cocaine. We provide evidence indicating that DA neurons are heterogeneous in their response to cocaine and that VTA non-DA neurons play an active role in processing systemic cocaine.

Role of glutamatergic projections from ventral tegmental area to lateral habenula in aversive conditioning.

The ventral tegmental area (VTA) plays roles in both reward and aversion. The participation of VTA in diverse behaviors likely reflects its heterogeneous neuronal phenotypes and circuits. Recent findings indicate that VTA GABAergic neurons that coexpress tyrosine hydroxylase (TH) projecting to lateral habenula (LHb) play a role in reward. In addition to these mesohabenular TH-GABAergic neurons, the VTA has many neurons expressing vesicular glutamate transporter 2 (VGluT2) that also project to LHb. To determine the behavioral role of mesohabenular VGluT2 neurons, we targeted channelrhodopsin2 to VTA VGluT2 neurons of VGluT2::Cre mice. These mice were tested in an apparatus where moving into one chamber stimulated VTA VGluT2 projections within the LHb, and exiting the chamber inactivated the stimulation. We found that mice spent significantly less time in the chamber where VGluT2 mesohabenular fiber stimulation occurred. Mice that received injections of mixed AMPA and NMDA glutamate receptor antagonists in LHb were unresponsive to VGluT2-mesohabenular fiber stimulation, demonstrating the participation of LHb glutamate receptors in mesohabenular stimulation-elicited aversion. In the absence of light stimulation, mice showed a conditioned place aversion to the chamber that was previously associated with VGluT2-mesohabenular fiber stimulation. We conclude that there is a glutamatergic signal from VTA VGluT2-mesohabenular neurons that plays a role in aversion by activating LHb glutamatergic receptors.

Single rodent mesohabenular axons release glutamate and GABA.

The lateral habenula (LHb) is involved in reward, aversion, addiction and depression through descending interactions with several brain structures, including the ventral tegmental area (VTA). The VTA provides reciprocal inputs to LHb, but their actions are unclear. Here we show that the majority of rat and mouse VTA neurons innervating LHb coexpress markers for both glutamate signaling (vesicular glutamate transporter 2; VGluT2) and GABA signaling (glutamic acid decarboxylase; GAD, and vesicular GABA transporter; VGaT). A single axon from these mesohabenular neurons coexpresses VGluT2 protein and VGaT protein and, surprisingly, establishes symmetric and asymmetric synapses on LHb neurons. In LHb slices, light activation of mesohabenular fibers expressing channelrhodopsin2 driven by VGluT2 (Slc17a6) or VGaT (Slc32a1) promoters elicits release of both glutamate and GABA onto single LHb neurons. In vivo light activation of mesohabenular terminals inhibits or excites LHb neurons. Our findings reveal an unanticipated type of VTA neuron that cotransmits glutamate and GABA and provides the majority of mesohabenular inputs.

Dopamine D4 receptor excitation of lateral habenula neurons via multiple cellular mechanisms.

Glutamatergic lateral habenula (LHb) output communicates negative motivational valence to ventral tegmental area (VTA) dopamine (DA) neurons via activation of the rostromedial tegmental nucleus (RMTg). However, the LHb also receives a poorly understood DA input from the VTA, which we hypothesized constitutes an important feedback loop regulating DA responses to stimuli. Using whole-cell electrophysiology in rat brain slices, we find that DA initiates a depolarizing inward current (I(DAi)) and increases spontaneous firing in 32% of LHb neurons. I(DAi) was also observed upon application of amphetamine or the DA uptake blockers cocaine or GBR12935, indicating involvement of endogenous DA. I(DAi) was blocked by D4 receptor (D4R) antagonists (L745,870 or L741,742), and mimicked by a selective D4R agonist (A412997). I(DAi) was associated with increased whole-cell conductance and was blocked by Cs+ or a selective blocker of hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel, ZD7288. I(DAi) was also associated with a depolarizing shift in half-activation voltage for the hyperpolarization-activated cation current (Ih) mediated by HCN channels. Recordings from LHb neurons containing fluorescent retrograde tracers revealed that I(DAi) was observed only in cells projecting to the RMTg and not the VTA. In parallel with direct depolarization, DA also strongly increased synaptic glutamate release and reduced synaptic GABA release onto LHb cells. These results demonstrate that DA can excite glutamatergic LHb output to RMTg via multiple cellular mechanisms. Since the RMTg strongly inhibits midbrain DA neurons, activation of LHb output to RMTg by DA represents a negative feedback loop that may dampen DA neuron output following activation.

Evidence for a regional specificity in the density and distribution of noradrenergic varicosities in rat cortex.

The brainstem nucleus locus coeruleus (LC) is the sole source of norepinephrine (NE)-containing fibers in the mammalian cortex. Previous studies suggest that the density of noradrenergic fibers in rat is relatively uniform across cortical regions and that cells in the nucleus discharge en masse. This implies that activation of the LC results in equivalent release of NE throughout the cortex. However, it is possible that there could be differences in the density of axonal varicosities across regions, and that these differences, rather than a difference in fiber density, may contribute to the regulation of NE efflux. Quantification of dopamine ß-hydroxylase (DßH)-immunostained varicosities was performed on several cortical regions and in the ventral posterior medial (VPM) thalamus by using unbiased sampling methods. The density of DßH varicosities is greater in the prefrontal cortex than in motor, somatosensory, or piriform cortices, greater in superficial than in deep layers of cortex, and greater in the VPM than in the somatosensory cortex. Our results provide anatomical evidence for non-uniform release of NE across functionally discrete cortical regions. This morphology may account for a differential, region-specific, impact of LC output on different cortical areas.

Adrenergic and noradrenergic innervation of the midbrain ventral tegmental area and retrorubral field: prominent inputs from medullary homeostatic centers.

Adrenergic agents modulate the activity of midbrain ventral tegmental area (VTA) neurons. However, the sources of noradrenergic and adrenergic inputs are not well characterized. Immunostaining for dopamine beta-hydroxylase revealed fibers within dopamine (DA) neuron areas, with the highest density in the retrorubral field (A8 cell group), followed by the VTA (A10 cell group), and very few fibers within substantia nigra compacta. A less dense, but a similar pattern of fibers was also found for the epinephrine marker, phenylethanolamine N-methyl transferase. Injection of the retrograde tracer wheat germ agglutinin-apo (inactivated) horseradish peroxidase conjugated to colloidal gold, or cholera toxin subunit b, revealed that the noradrenergic innervation of the A10 and A8 regions arise primarily from A1, A2, A5, and locus ceruleus neurons. Selective lesions of the ventral noradrenergic bundle confirmed a prominent innervation from A1 and A2 areas. Retrogradely labeled epinephrine neurons were found mainly in the C1 area. The identification of medullary noradrenergic and adrenergic afferents to DA neuron areas indicates new pathways for visceral-related inputs to reward-related areas in the midbrain.

Sex differences in models of temporal lobe epilepsy: role of testosterone.

Kainic acid and pilocarpine were used to assess sex differences in temporal lobe seizures. Adult Sprague-Dawley rats were injected with kainic acid (10-12 mg/kg) or with pilocarpine (380 mg/kg) and behavior was recorded for the next 3 h. Trunk blood was collected for hormonal measurements. Our data indicate that the male is more susceptible to the convulsant effects of agents that produce temporal lobe-like seizures. Males presented a higher amount of full limbic convulsions than females. To assess the role of plasma testosterone levels in kainate-induced seizures, a group of males was gonadectomized and half received testosterone replacement. The presence of testosterone, in intact and in gonadectomized males with testosterone replacement, increased the susceptibility to seizure. Seizures were either stronger (full limbic) or more frequent in animals with testosterone compared to animals devoid of testosterone. These results suggest that differences in plasma levels of testosterone may be partially responsible for the observed gender differences in seizure susceptibility. Our data reveal a reciprocal relationship between kainic acid-induced temporal lobe seizures and plasma testosterone. Testosterone enhances the occurrence and the severity of seizures. Conversely, kainic-acid-induced seizures decrease plasma testosterone. The higher plasma corticosterone levels found in these males suggest that kainic acid-induced seizures activate the hypothalamic-pituitary-adrenal axis which may induce alterations in plasma levels of male reproductive hormones.