Neurotrophic effects of serum- and glucocorticoid-inducible kinase on adult murine mesencephalic dopamine neurons.

Mesencephalic dopamine neurons are central to many aspects of human cognition, motivational, and motor behavior, and they are uniquely vulnerable to degenerative neurologic disorders such as Parkinson's disease. There is growing evidence that in the mature brain these neurons not only remain responsive to neurotrophic support, but are dependent on it for viability and function. Little is known of the cellular signaling pathways that mediate this support, although some evidence suggests that protein kinase Akt/PKB may play such a role. Another candidate for such a role is serum- and glucocorticoid-inducible kinase (SGK), a member of the AGC kinase family that is closely related to Akt. We have herein examined the responsiveness of adult mouse dopamine neurons in vivo to overexpression of wild-type and a constitutively active form of SGK by use of viral vector transfer in normal mice and both before and after 6-OHDA lesion. We find that SGK induces a broad spectrum of neurotrophic effects on these neurons, including induction of neuronal hypertrophy, protection from both neuron death and neurotoxin-induced retrograde axonal degeneration, and axon regeneration. Given the diverse and robust effects of SGK on these neurons, and its abundant expression in them, we suggest that SGK, like closely related Akt, may play a role in their responsiveness to neurotrophic factors and in adult maintenance. It therefore offers a novel target for therapeutic development.

Selective anterograde tracing of nonserotonergic projections from dorsal raphe nucleus to the basal forebrain and extended amygdala.

The dorsal raphe nucleus (DRN) contains both serotonergic and nonserotonergic projection neurons. Retrograde tracing studies have demonstrated that components of the basal forebrain and extended amygdala are targeted heavily by input from nonserotonergic DRN neurons. The object of this investigation was to examine the terminal distribution of nonserotonergic DRN projections in the basal forebrain and extended amygdala, using a technique that allows selective anterograde tracing of nonserotonergic DRN projections. To trace nonserotonergic DRN projections, animals were pretreated with nomifensine, desipramine and the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), 7 days prior to placing an iontophoretic injection of biotinylated dextran amine (BDA) into the DRN. In animals treated with 5,7-DHT, numerous nonserotonergic BDA-labeled fibers ascended to the basal forebrain in the medial forebrain bundle system. Some of these labeled fibers crossed through the lateral hypothalamus, bed nucleus of the stria terminalis, and substantial innominata. These fibers entered the amygdala through the ansa peduncularis and ramified within the central and basolateral amygdaloid nuclei. Other fibers entered the diagonal band of Broca and formed a dense plexus of labeled fibers in the dorsal half of the intermediate portion of the lateral septal nucleus and the septohippocampal nucleus. These findings demonstrate that the basal forebrain and extended amygdala receive a dense projection from nonserotonergic DRN neurons. Given that the basal forebrain plays a critical role in processes such as motivation, affect, and behavioral control, these findings support the hypothesis that nonserotonergic DRN projections may exert substantial modulatory control over emotional and motivational functions.

Neonatal serotonin depletion alters behavioral responses to spatial change and novelty.

Multiple brain disorders that show serotonergic imbalances have a developmental onset. Experimental models indicate a role for serotonin as a morphogen in brain development. To selectively study the effects of serotonin depletions on cortical structural development and subsequent behavior, we developed a mouse model in which a serotonin neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), is injected into the medial forebrain bundle (mfb) on the day of birth. Littermates with saline injections into the mfb and age matched mice served as controls. This study characterized the extent and duration of serotonergic denervation after the selective neonatal lesion and investigated effects on exploratory behavior, spatial learning and anxiety in mice of both sexes. We report significant decreases in the serotonergic (5-HT) innervation to cortex and hippocampus, but not to subcortical forebrain structures in 5,7-DHT-lesioned mice. The depletion of 5-HT fibers in cortical areas was long lasting in lesioned mice but autoradiographic binding to high affinity 5-HT transporters was only transiently reduced. Male but not female lesioned mice reduced their exploration significantly in response to spatial rearrangement and object novelty, suggesting increased anxiety in response to change but normal spatial cognition. Our data show that developmental disruptions in the serotonergic innervation of cortex and hippocampus are sufficient to induce permanent, sex specific, behavioral alterations. These results may have significant implications for understanding brain disorders presenting with cortical morphogenetic abnormalities and altered serotonin neurotransmission, such as autism, schizophrenia and affective disorders.

1,2,3,4-Tetrahydroisoquinoline protects terminals of dopaminergic neurons in the striatum against the malonate-induced neurotoxicity.

Malonate, a reversible inhibitor of the mitochondrial enzyme succinate dehydrogenase, is frequently used as a model neurotoxin to produce lesion of the nigrostriatal dopaminergic system in animals due to particular sensitivity of dopamine neurons to mild energy impairment. This model of neurotoxicity was applied in our study to explore neuroprotective potential of 1,2,3,4-tetrahydroisoquinoline (TIQ), an endo- and exogenous substance whose function in the mammalian brain, despite extensive studies, has not been elucidated so far. Injection of malonate at a dose of 3 mumol unilaterally into the rat left medial forebrain bundle resulted in the 54% decrease in dopamine (DA) concentration in the ipsilateral striatum and, depending on the examined striatum regions, caused 24-44% reduction in [3H]GBR12,935 binding to the dopamine transporter (DAT). TIQ (50 mg/kg i.p.) administered 4 h before malonate infusion and next once daily for successive 7 days prevented both these effects of malonate. Such TIQ treatment restored DA content and DAT binding almost to the control level. The results of the present study indicate that TIQ may act as a neuroprotective agent in the rat brain. An inhibition of the enzymatic activities of monoamine oxidase and gamma-glutamyl transpeptidase as well as an increase in the striatal levels of glutathione and nitric oxide found after TIQ administration and reported in our earlier studies are considered to be potential factors that may be involved in the TIQ-mediated protection of dopamine terminals from malonate toxicity.

Median bundle neurons coordinate behaviours during Drosophila male courtship.

Throughout the animal kingdom the innate nature of basic behaviour routines suggests that the underlying neuronal substrates necessary for their execution are genetically determined and developmentally programmed. Complex innate behaviours require proper timing and ordering of individual component behaviours. In Drosophila melanogaster, analyses of combinations of mutations of the fruitless (fru) gene have shown that male-specific isoforms (Fru(M)) of the Fru transcription factor are necessary for proper execution of all steps of the innate courtship ritual. Here, we eliminate Fru(M) expression in one group of about 60 neurons in the Drosophila central nervous system and observe severely contracted courtship behaviour, including rapid courtship initiation, absence of orienting and tapping, and the simultaneous occurrence of wing vibration, licking and attempted copulation. Our results identify a small group of median bundle neurons, that in wild-type Drosophila appropriately trigger the sequential execution of the component behaviours that constitute the Drosophila courtship ritual.

Activation of protein kinase C by the error signal from a basal ganglia-forebrain circuit in the zebra finch song control nuclei.

An error signal from the anterior forebrain pathway (AFP) in the songbird brain is necessary for juvenile song learning and adult song maintenance. It induces the expression of protein kinase C (PKC) which is related to the plasticity in the robust nucleus of the archistriatum (RA), one of the song control nuclei in the forebrain. The glutamatergic inputs from the AFP activate mainly the NMDA receptors of the RA neurons. In order to clarify the molecular mechanism of error signal-induced PKC activation, two experiments were carried out. First, Ca2+ concentration was measured in a brain slice preparation from zebra finches using the fluorescent Ca2+ indicator Fura 2-AM. Glutamate increased the intracellular Ca2+ concentration ([Ca2+](i)) in RA neurons. This increase was inhibited by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (AP5). Second, we examined the expression of PKC in the RA slice preparation after stimulation with glutamate for 10 min using PKCbeta1 fluorescence immunohistochemistry. Glutamate induced the activation of PKC as the translocation from the cytosol to the cell membrane, and the translocation was inhibited by AP5. These results indicate that the translocation of the PKC caused by the [Ca2+](i) elevation through NMDA receptors is concerned with the initial stage of error signal-induced plasticity in the RA.

Adeno-associated viral delivery of GDNF promotes recovery of dopaminergic phenotype following a unilateral 6-hydroxydopamine lesion.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for dopamine neurons that has been proposed for use in the treatment of Parkinson's disease (PD). Previous studies using viral vectors to deliver GDNF in rodent models of PD have entailed administering the virus either prior to or immediately after neurotoxin-induced lesions, when the nigrostriatal pathway is largely intact, a paradigm that does not accurately reflect the clinical situation encountered with Parkinson's patients. In this study, recombinant adeno-associated virus carrying the gene encoding GDNF (rAAV-GDNF) was administered to animals bearing a maximal lesion in the nigrostriatal system, more closely resembling fully developed PD. Rats were treated with 6-hydroxydopamine into the medial forebrain bundle and assessed by apomorphine-induced rotational behavior for 5 weeks prior to virus administration. Within 4 weeks of a single intrastriatal injection of rAAV-GDNF, unilaterally lesioned animals exhibited significant behavioral recovery, which correlated with increased expression of dopaminergic markers in the substantia nigra, the medial forebrain bundle, and the striatum. Our findings demonstrate that rAAV-GDNF is capable of rescuing adult dopaminergic neurons from near complete phenotypic loss following a neurotoxic lesion, effectively restoring a functional dopaminergic pathway and diminishing motoric deficits. These data provide further support for the therapeutic potential of rAAV-GDNF-based gene therapy in the treatment of PD.

Basic organization of projections from the oval and fusiform nuclei of the bed nuclei of the stria terminalis in adult rat brain.

The organization of axonal projections from the oval and fusiform nuclei of the bed nuclei of the stria terminalis (BST) was characterized with the Phaseolus vulgaris-leucoagglutinin (PHAL) anterograde tracing method in adult male rats. Within the BST, the oval nucleus (BSTov) projects very densely to the fusiform nucleus (BSTfu) and also innervates the caudal anterolateral area, anterodorsal area, rhomboid nucleus, and subcommissural zone. Outside the BST, its heaviest inputs are to the caudal substantia innominata and adjacent central amygdalar nucleus, retrorubral area, and lateral parabrachial nucleus. It generates moderate inputs to the caudal nucleus accumbens, parasubthalamic nucleus, and medial and ventrolateral divisions of the periaqueductal gray, and it sends a light input to the anterior parvicellular part of the hypothalamic paraventricular nucleus and nucleus of the solitary tract. The BSTfu displays a much more complex projection pattern. Within the BST, it densely innervates the anterodorsal area, dorsomedial nucleus, and caudal anterolateral area, and it moderately innervates the BSTov, subcommissural zone, and rhomboid nucleus. Outside the BST, the BSTfu provides dense inputs to the nucleus accumbens, caudal substantia innominata and central amygdalar nucleus, thalamic paraventricular nucleus, hypothalamic paraventricular and periventricular nuclei, hypothalamic dorsomedial nucleus, perifornical lateral hypothalamic area, and lateral tegmental nucleus. Moderately dense inputs are found in the parastrial, tuberal, dorsal raphé, and parabrachial nuclei and in the retrorubral area, ventrolateral division of the periaqueductal gray, and pontine central gray. Light projections end in the olfactory tubercle, lateral septal nucleus, posterior basolateral amygdalar nucleus, supramammillary nucleus, and nucleus of the solitary tract. These and other results suggest that the BSTov and BSTfu are basal telencephalic parts of a circuit that coordinates autonomic, neuroendocrine, and ingestive behavioral responses during stress.

Responses of ventral tegmental area GABA neurons to brain stimulation reward.

Dopamine neurons in the ventral tegmental area (VTA) have been implicated in rewarded behaviors, including intracranial self-stimulation (ICSS). We demonstrate, in unrestrained rats, that the discharge activity of a homogeneous population of presumed VTA GABA neurons, implicated in cortical arousal, increases before ICSS of the medial forebrain bundle (MFB). These findings suggest that VTA GABA neurons may be involved in the attentive processes related to brain stimulation reward (BSR).

Relapse to cocaine-seeking after hippocampal theta burst stimulation.

Treatment efforts for cocaine addiction are hampered by high relapse rates. To map brain areas underlying relapse, we used electrical brain stimulation and intracranial injection of pharmacological compounds after extinction of cocaine self-administration behavior in rats. Electrical stimulation of the hippocampus containing glutamatergic fibers, but not the medial forebrain bundle containing dopaminergic fibers, elicited cocaine-seeking behavior dependent on glutamate in the ventral tegmental area. This suggests a role for glutamatergic neurotransmission in relapse to cocaine abuse. The medial forebrain bundle electrodes supported intense electrical self-stimulation. These findings suggest a dissociation of neural systems subserving positive reinforcement (self-stimulation) and incentive motivation (relapse).

Release and elimination of dopamine in vivo in mice lacking the dopamine transporter: functional consequences.

In mice lacking the dopamine transporter (DAT), the amplitude of dopamine (DA) release and the kinetics of dopamine elimination were measured in vivo using carbon fibre electrodes combined with amperometry. DA release was evoked by electrical stimulation of the medial forebrain bundle. The amplitude of DA release per pulse was lower (7% in striatum and 21% in nucleus accumbens) than in wild-type mice. Inhibition of monoamine oxidases (MAOs) by pargyline, but not of catechol-O-methyltransferase (COMT) by tolcapone, slowed down DA elimination in knockout mice. As DA half-life was two orders of magnitude higher in these mice, the DA diffusion distance was 10-times higher than in wild-types (100 and 10 microm, respectively). In knockout mice, alpha-methyl-p-tyrosine induced a much faster decline of DA release and haloperidol was less effective in potentiating DA release. Therefore, DA release was more dependent on DA synthesis than in normal animals but was less influenced by D2 autoregulation. Dopaminergic neurons exhibit two kinds of discharge activity, i.e. single spikes and bursts of 2-6 action potentials. In wild-type mice, stimuli mimicking bursts evoked significant increases in extracellular DA over its basal level sustained by tonic activity. However, in mice lacking the DAT, low frequency firing resulted in consistently high extracellular DA levels that could not be distinguished from DA levels achieved by high frequency firing. Therefore, the burst firing activity cannot be specifically translated into phasic changes in extracellular DA. This deficit might contribute to the difficulties of these mice in spatial cognitive function.

Multisecond oscillations in the subthalamic nucleus: effects of apomorphine and dopamine cell lesion.

Clinical and preclinical data indicate that the subthalamic nucleus (STN) plays a critical role in mediating the hyper- and hypoactive behavioral states associated with increases and decreases in dopamine receptor stimulation in the basal ganglia. The present study investigates effects of dopamine receptor stimulation on slow multisecond oscillations in firing rates in STN neurons. Extracellular, single-unit recordings were performed in locally anesthetized and immobilized rats which were either intact or had received unilateral 6-OHDA lesions of the medial forebrain bundle. The majority (64%) of spike trains recorded from STN neurons exhibited periodic oscillations in firing rate within the range of 2-60 sec, with an average period of 24 sec. The distribution of these baseline periodicities was not altered by unilateral 6-OHDA lesion, but periods were significantly shortened by systemic administration of the D1/D2 agonist apomorphine. This effect was observed in a greater proportion of neurons recorded from 6-OHDA-lesioned rats as compared to intact rats, was notably diminished in rats systemically anesthetized with chloral hydrate, and did not correlate with drug-induced changes in firing rate. These oscillations are similar to slow periodicities in firing rate recently reported in other basal ganglia nuclei. The possibility that these periodic oscillations in firing rate play a significant role in basal ganglia function was supported by the observation that the time of onset of apomorphine induced alterations in amplitude and periodicity of slow oscillations in STN spike trains is coincident with the onset of behavioral effects of this drug in 6-OHDA-lesioned animals. Synapse 38:38-50, 2000. Published 2000 Wiley-Liss, Inc.

A projection from the ventral tegmental area to the periaqueductal gray involved in cardiovascular regulation.

Experiments were done in alpha-chloralose-anesthetized rats to determine a pathway mediating the cardiovascular depressor responses elicited from stimulation of the ventral tegmental area (VTA). The magnitude of the depressor responses elicited by glutamate stimulation (0.1 M/30 nl) of the VTA was examined after neuronal block produced by microinjections of lidocaine into ascending fiber bundles leaving the VTA to innervate the forebrain and thalamus. Bilateral microinjections of 1 microl of 4% lidocaine in the medial forebrain bundle (n = 6) and in the periventricular fibers of the midbrain (n = 5) did not attenuate the depressor response from stimulation of the VTA. Experiments were done using the anterograde tracer biotinylated dextran amine to identify descending projections from the VTA to cardiovascular centers in the brain stem. Examination of the nucleus of the solitary tract, ventrolateral medulla, and A5 catecholaminergic cell group revealed few or no fibers or terminals. Occasional fibers and some terminals were observed in the nucleus of raphe magnus, parabrachial nucleus, and locus ceruleus. A very dense bilateral projection was found to the ventrolateral periaqueductal gray (PAGvl) and dorsal raphe nucleus adjacent to the PAGvl. Bilateral injections of 4% lidocaine (n = 4) or 10 mM cobalt chloride (n = 5) into the PAGvl region attenuated the depressor responses elicited by stimulation of the VTA by approximately 50%. These experiments indicate that the depressor responses elicited from activation of the VTA are mediated in part by a pathway to a cardiovascular depressor area located in the PAGvl.

Organization of projections from the juxtacapsular nucleus of the BST: a PHAL study in the rat.

The axonal projections of the juxtacapsular nucleus of the anterior division of the bed nuclei of the stria terminalis (BSTju) were examined with the Phaseolus vulgaris-leucoagglutinin (PHAL) method in the adult male rat. Our results indicate that the BSTju displays a relatively simple projection pattern. First, it densely innervates the medial central amygdalar nucleus and the subcommissural zone and caudal anterolateral area of the BST - cell groups involved in visceromotor responses. Second, it provides inputs to the ventromedial caudoputamen (CP) and anterior basolateral amygdalar nucleus - areas presumably modulating somatomotor outflow. Third, the BSTju sends dense projections to the caudal substantia innominata, a distinct caudal dorsolateral region of the compact part of the substantia nigra, and the adjacent mesencephalic reticular nucleus and retrorubral area. And fourth, the BSTju provides light inputs to the prelimbic, infralimbic, and ventral CA1 cortical areas; to the posterior basolateral, posterior basomedial, and lateral amygdalar nuclei; to the paraventricular and medial mediodorsal thalamic nuclei; to the subthalamic and parasubthalamic nuclei of the hypothalamus; and to the ventrolateral periaqueductal gray. These projections, in part, suggest a role for the BSTju in circuitry integrating autonomic responses with somatomotor activity in adaptive behaviors.

Serotonin hyperinnervation in the adult rat ventral mesencephalon following unilateral transection of the medial forebrain bundle. Correlation with reactive microglial and astroglial populations.

We have previously studied changes in the serotoninergic and dopaminergic nigrostriatal systems following transection of the medial forebrain bundle and found a long-term axotomy-induced increase in the levels of serotonin and its main metabolite, 5-hydroxyindolacetic acid in substantia nigra [Venero et al. (1997) J. Neurochem. 68, 2458-2468]. In an attempt to find a rationale for this effect, we have performed an immunohistochemical study. Transection of the medial forebrain bundle of the rat interrupted most of the ascending serotoninergic pathways from the raphe nuclei as revealed by serotonin immunoreactivity. While serotonin immunostaining was almost absent in striatum, it doubled in the ventral mesencephalon at 21 days postlesion. This axotomy-induced increase was accompanied by an increased density of the serotonin nerve terminal network in the ipsilateral substantia nigra and ventral tegmental area. The increase in serotonin immunoreactivity was in line with the measured levels of serotonin and 5-hydroxyindolacetic acid in substantia nigra. In addition, the distribution pattern of glial fibrillary acidic protein-immunoreactive astrocytes and OX42-immunoreactive microglia correlated highly with the location of increased serotonin fibre density in the ventral mesencephalon, especially in ventral tegmental area and in the most medial part of substantia nigra. We suggest that a pruning effect may underly the axotomy-induced increase in serotonin immunoreactivity in the ventral mesencephalon, and further, that activated astroglia and microglia may play a role in directing serotoninergic axonal regeneration following axotomy.

Angiotensin II receptor binding sites in the ventral portion of the bed nucleus of the stria terminalis are reduced by interruption of the medial forebrain bundle.

Many techniques have been utilized to discern the localization of angiotensin II (Ang II) receptors to specific cellular components (glia, neuronal cell bodies and nerve terminals) in the brain. In the present study, we used lesioning techniques to localize Ang II receptors to cellular components in the rat forebrain. In the first experiment, axons ascending to the hypothalamus and forebrain from neurons in the brainstem were destroyed by unilaterally cutting the medial forebrain bundle (MFB). In the second experiment, a single injection of the neurotoxin, ibotenic acid, was injected unilaterally into the ventral portion of the bed nucleus of the stria terminalis (BSTV) to destroy neuronal cell bodies, thus determining if Ang II receptors are present on neuronal cell bodies. In both experiments, the animals were sacrificed after two weeks recovery and the brains processed for in vitro receptor autoradiography using 125I-sar1,ile8 Ang II (125I-SI Ang II). Unilateral knife-cut lesions of the MFB caused a significant reduction in 125I-SI Ang II binding in the BSTV (30+/-6%) and the piriform cortex (PC; 26+/-4%) ipsilateral to the knife cut. Unilateral injection of the neurotoxin into the BSTV failed to alter 125I-SI Ang II binding in this nucleus. These experiments suggest that at least a subpopulation of Ang II receptors in the BSTV and PC are located on terminals of neurons that have their cell bodies in the brainstem and their axons in the MFB.

Coupled effects of mass transfer and uptake kinetics on in vivo microdialysis of dopamine.

Voltammetric microelectrodes and microdialysis probes were used simultaneously to monitor extracellular dopamine in rat striatum during electrical stimulation of the medial forebrain bundle. Microelectrodes were placed far away (1 mm) from, immediately adjacent to, and at the outlet of microdialysis probes. In drug-naive rats, electrical stimulation (45 Hz, 25 s) evoked a robust response at microelectrodes far away from the probes, but there was no response at microelectrodes adjacent to and at the outlet of the probes. After nomifensine administration (20 mg/kg i.p.), stimulation evoked robust responses at all three microelectrode placements. These results demonstrate first that evoked release in tissue adjacent to microdialysis probes is suppressed in comparison with evoked release in tissue far away from the probes and second that equilibration of the dopamine concentration in the extracellular fluid adjacent to and far away from the probes is prevented by the high-affinity dopamine transporter. Hence, models of microdialysis, which assume the properties of tissue to be spatially uniform, require modification to account for the distance that separates viable sites of evoked dopamine release from the probe. We introduce new mass transfer resistance parameters that qualitatively explain the observed effects of uptake inhibition on stimulation responses recorded with microdialysis and voltammetry.

Unilateral lesion of dorsal hippocampus enhances reinforcing lateral hypothalamic stimulation in the contralateral hemisphere.

Whereas convincing evidence exists for an important role of the hippocampus in mechanisms underlying memory and encoding of location in space, the contribution of the hippocampus to the system underlying central processes of reinforcement is less well established. Scattered data suggesting that hippocampal ablation increases the effectiveness of positive reinforcers have alternatively been interpretated in terms of general and unspecific behavioral disinhibition, which results in higher levels of activity and rates of responding. In the present experiment, 22 Wistar rats were either given a neurotoxic or a sham lesion in the CA1 region of the hippocampus, and the effect on lateral hypothalamic self-stimulation behavior was assessed. To control for nonspecific performance effects rates of lever pressing were assessed ipsi- and contralateral to the lesioned hemisphere as well as under condition of extinction (current set to zero). Following the neurotoxic lesion the animals displayed significant higher rates of self-stimulation at the electrode sites in the hypothalamus situated contralateral but not ipsilateral to the hemisphere with the lesion compared with controls. The increase in self-stimulation commenced on the third day postlesion and was maintained over the 8 days of testing. The lesion did not change the animals' behavior under extinction. Thus, the hippocampal lesion led to an amplification of rewarding lateral hypothalamic self-stimulation behavior, indicative of a lesion induced disinhibition of the brain's reinforcement system.

Ipsilateral alterations in tryptophan hydroxylase activity in rat brain after hypothalamic 5,7-di-hydroxytryptamine lesion.

The in vivo relationship between the amounts of tryptophan hydroxylase (TPH) protein and its intrinsic synthetic activity, measured by quantifying the amounts of alpha-[3H]methyl-5-hydroxytryptamine (alpha-[3H]M5-HT), is reported in cell body and terminal areas of intact and disturbed serotonergic neurons following a unilateral 5,7-dihydroxytryptamine (5,7-DHT) lesion of the dorsolateral hypothalamus. Five days after the lesion, the relationships between TPH and its synthetic product 5-HT were evaluated on adjacent brain sections in serotonergic cells bodies of the dorsal raphe nucleus (DRN) and nerve fibres of the medial forebrain bundle (MFB). On the side contralateral to the lesion, TPH and alpha-[3H]M5-HT levels in the intact hemi-DRN exhibited a caudo-rostral distribution and were positively and significantly correlated (p < or = 0.001); the calculated TPH-specific activity was 0.76 nCi of alpha-[3H]M5-HT formed per U TPH. In the MFB, quantitative measurements of TPH and alpha-[3H]M5-HT showed no correlation between enzyme and product and no specific activity for TPH could be determined. On the side ipsilateral to the lesion, the density of TPH-immunoreactive fibers was drastically decreased in the dorsolateral hypothalamus where a significant reduction in TPH content (45.5% of control side, P < 0.001) was found. In the overall ipsilateral hemi-DRN, TPH and alpha-[3H]M5-HT levels, their correlation as well as TPH-specific activity were unaltered by the lesion but a significant increase in alpha-[3H]M5-HT and TPH contents was observed in the lateral wings of the DRN. The lesion also induced a significant increase in alpha-[3H]M5-HT and TPH levels (136% and 93.8%, P < 0.001, respectively) in the ipsilateral MFB, which resulted in a positive and significant correlation between these two markers and yielded a TPH-specific activity of 1.0 nCi of alpha-[3H]M5-HT formed per U TPH. TPH topological area was also significantly increased in the lateral aspect of the ipsilateral MFB 5 days post lesion. These results show that 5-HT synthesis in the intact DRN is proportional to and dependent on TPH activity while in the MFB, 5-HT accumulation appears unrelated to TPH content which is most likely in an inactive enzymatic form. Moreover, the data show that a local disruption of serotonergic terminals in the dorsolateral hypothalamus does not affect 5-HT synthesis in the overall ipsilateral DRN neurons but results in local activation of TPH within the serotonergic projection neurons and the ipsilateral MFB, as evidenced by active de novo synthesis of 5-HT. Altogether the results point to circumscribed activation of compensatory mechanisms in 5-HT synthesis after selective destruction of serotonergic terminals.

Increased ipsilateral expression of Fos following lateral hypothalamic self-stimulation.

Immunohistochemical labeling of Fos protein was used to visualize neurons activated by rewarding stimulation of the lateral hypothalamic level of the medial forebrain bundle (MFB). Following training and stabilization of performance, seven rats were allowed to self-stimulate for 1 h prior to anesthesia and perfusion. Brains were then processed for immunohistochemistry. Two control subjects were trained and tested in an identical manner except that the stimulator was disconnected during the final 1 h test. Among the structures showing a greater density of labeled neurons on the stimulated side of the brains of the experimental subjects were the septum, lateral preoptic area (LPO), medial preoptic area, bed nucleus of the stria terminalis, substantia innominata (SI), and the lateral hypothalamus (LH). Several of these structures, the LPO, SI, and LH, have been implicated in MFB self-stimulation by the results of psychophysical, electrophysiological, and lesion studies.