targeted-toxins

Li A-J, Dinh TT, Ritter S (2007) Destruction of NPY receptor expressing neurons in the arcuate nucleus causes obesity and hyperphagia without increasing lateral hypothalamic orexigenic peptide gene expression. Neuroscience 2007 Abstracts 524.20/BBB20. Society for Neuroscience, San Diego, CA.

Summary: NPY-SAP, a conjugate of neuropeptide Y (NPY) and the ribosomal inactivating toxin, saporin (SAP), is a compound that selectively lesions NPY receptor-expressing neurons. Previously we showed that injection of NPY-SAP into the hypothalamic arcuate nucleus (ARC) induces hyperphagia and obesity in rats. To further investigate the mechanisms responsible for NPY-SAP-induced obesity, we injected NPY-SAP or blank-saporin (B-SAP) control into the ARC and subsequently examined the expression of two orexigenic neuropeptide genes in the lateral hypothalamic area (LHA), which is densely innervated by ARC neurons. Our hypothesis was that loss of leptin-sensitive neurons in the ARC in the NPY-SAP injected rats would lead to increased expression of orexigenic neurons elsewhere in the hypothalamic feeding circuitry. Body weight gain and food intake were dramatically increased in the NPY-SAP group. In addition, expression of NPY and cocaine- and amphetamine-regulated transcript (CART) mRNA was significantly reduced in the ARC of obese rats, indicating a loss of NPY receptor-expressing NPY and CART neurons in this region. In contrast, NPY and CART gene expression in the dorsomedial hypothalamic nucleus was unchanged in NPY-SAP rats, indicating that the NPY-SAP-induced lesion was limited to the ARC. However, contrary to our hypothesis, expression of the orexigenic neuronpeptides, melanin-concentrating hormone (MCH) or prepro-orexin mRNA in LHA was not enhanced, but was slightly reduced in the NPY-SAP rats. These results indicate that an enhancement of MCH or orexin expression in the LHA is not necessary for the hyperphagia and obesity observed after NPY-SAP lesions in the ARC. Supported by PHS grant #DK 40498.

Related Products: NPY-SAP (Cat. #IT-28), Blank-SAP (Cat. #IT-21)

Haller J, Toth M, Zelena D, Halasz J (2007) Molecular basis of violent behavior: The role of NK1 receptors. Neuroscience 2007 Abstracts 531.22/GGG24. Society for Neuroscience, San Diego, CA.

Summary: Background. Neurons expressing Neurokinin1 receptor (NK1 or Substance P receptor) are abundant in limbic areas crucial for different emotional behaviors. In recent years, NK1 receptor blockers were proposed for the treatment of anxiety and depression. Moreover, in two different laboratory models, NK1 receptor blockade was successfully used to decrease violent components of aggression related behaviors in Wistar rats (Biol. Psychiatry, 2007, in press). In the above study, the NK1 receptor blockade reduced the number of more violent hard bites, while the number of soft bites was unaltered. Aggressive encounters were accompanied by a marked activation of neurons expressing NK1 receptors in the medial amygdala and in the hypothalamic attack area, where the highest number and proportion of activated NK1 positive neurons were found. Aim / Methods. We evaluated the precise role of neurons expressing NK1 receptors in the hypothalamic attack area during resident/intruder test. These neurons were selectively eliminated by a Substance P conjugated saporin bilateral microinjection into the hypothalamic attack area. After a week recovery, lesioned and vehicle treated control residents were faced with a smaller untreated opponent in their home cages for 20 min. The brains of the residents were later removed to assess the site of injection and the extent of the lesion. Results. In lesioned Wistars, the bilateral microinjection resulted in a complete and selective disruption of NK1 positive neurons in the hypothalamic attack area. Compared to vehicle injected controls, the number of hard bites toward unfamiliar residents showed a marked decrease (almost a complete abolition) in the lesioned group. The latency of hard bites was significantly increased compared to vehicle injected controls. The number of bite attacks was also reduced, but this reduction was mainly secondary to the dramatic reduction in the number of hard bites. Conclusions. Our data show that hypothalamic neurons expressing NK1 receptors are involved in the control of aggressiveness, especially in the expression of violent attacks. These data confirm and support earlier results that NK1 antagonists – beyond anxiety and depression – may also be useful in the treatment of aggressiveness and violence.

Related Products: SP-SAP (Cat. #IT-07)

Gaffan D, Baxter MG, Browning PGF (2007) Intact delayed nonmatching-to-sample in monkeys with combined lesions of the temporal cortical cholinergic system and the fornix. Neuroscience 2007 Abstracts 341.11. Society for Neuroscience, San Diego, CA.

Summary: Rhesus monkeys were pre-operatively trained in truly trial-unique delayed nonmatching-to-sample (DNMS) in an automated apparatus. They were then divided into a control group (n=3) and an experimental group (n=3) and received injections into the inferior temporal cortex of either saline (controls) or the selective cholinergic immunotoxin ME20.4-saporin (experimentals). A postoperative DNMS test showed no significant impairment in the experimental group, both groups performing at their pre-operative level. Both groups then underwent a second surgery to transect the fornix. Again, there was no significant impairment in DNMS, both groups performing at their pre-operative level. If the lesions are confirmed histologically then these results are in marked contrast to our findings with scene learning, in which monkeys with the same combined lesion as those in the present experimental group were severely impaired. However, a number of recent studies have shown that tasks with temporally complex events extended over trials, like DNMS, discrimination learning set, and serial reversal set, depend on a short-term prospective memory strategy that is supported by the interaction of temporal cortex with prefrontal cortex. Thus, the performance of DNMS does not require the laying down of new long-term memories.

Related Products: ME20.4-SAP (Cat. #IT-15)

Browning PG, Gaffan D, Baxter MG (2007) Severe visual learning impairments in monkeys with combined but not separate lesions of the temporal cortical cholinergic system and the fornix. Neuroscience 2007 Abstracts 341.7. Society for Neuroscience, San Diego, CA.

Summary: A dense amnesia can be produced in the monkey by sectioning the anterior temporal stem, amygdala and fornix, a procedure which deafferents temporal cortex from modulatory inputs from the midbrain and basal forebrain. The present experiment investigated the neurochemical specificity of these severe learning impairments by selectively destroying cholinergic projections to the entire inferior temporal cortex by making multiple injections of the immunotoxin ME20.4-saporin into the inferior temporal cortex bilaterally. Six male macaque monkeys were preoperatively trained to learn new object-in-place discrimination problems each day until they could rapidly learn many such problems within a testing session. The monkeys then underwent surgery and received either injections of immunotoxin (n=3) or injections of saline (n=3). Both groups of monkeys were unimpaired when postoperative and preoperative performance were compared. Each monkey then underwent a second surgery to transect the fornix. After this surgery monkeys who had previously received injections of immunotoxin into temporal cortex showed a severe learning impairment, whereas monkeys who had previously received injections of saline showed a mild impairment. Monkeys with the combined immunotoxin plus fornix lesion were also severely impaired at concurrent object discrimination learning. These results suggest that different neuromodulatory inputs to inferior temporal cortex may act in concert to support cortical plasticity in visual learning such that the loss of acetylcholine only is not sufficient to disrupt normal learning behavior. The results also suggest that in monkeys, as in humans with Alzheimer’s disease, severe memory impairments occur only when a loss of acetylcholine projections to cortex is accompanied by organic tissue damage.

Related Products: ME20.4-SAP (Cat. #IT-15)

Baxter MG, Kyriazis DA, Croxson PL (2007) Cholinergic depletion of prefrontal cortex does not impair episodic memory or strategy implementation in rhesus monkeys. Neuroscience 2007 Abstracts 341.9. Society for Neuroscience, San Diego, CA.

Summary: The prefrontal cortex is involved in regulating multiple aspects of memory, decision-making, and cognitive control. Cholinergic input to prefrontal cortex is thought to be involved in supporting its functions. To examine this hypothesis we tested 4 rhesus monkeys (3 male) with cholinergic depletion of ventrolateral prefrontal cortex (N=2) or the entire prefrontal cortex, excluding its medial aspect (N=2). Selective cholinergic depletion was produced by multiple injections of the immunotoxin ME20.4-saporin (0.02 ug/ul) into the prefrontal cortex. These monkeys were tested on two tasks that each require frontal-inferotemporal interaction, as well as an intact ventrolateral prefrontal cortex. The first, strategy implementation, requires monkeys to apply different choice strategies to different categories of objects in order to maximize the rate of reward delivery, and engages decision-making and cognitive control. The second, scene memory, is a test of episodic memory in which monkeys rapidly learn 20 new object-in-place scene discrimination problems within a single test session. Cholinergic depletions of prefrontal cortex, whether they were limited to ventrolateral prefrontal cortex or included the whole of lateral and orbital prefrontal cortex, were without effect on either strategy implementation or new scene learning relative to each monkey’s preoperative performance. Thus, episodic memory and strategy implementation can proceed normally even with severely disrupted cholinergic input, so loss of cholinergic input on its own cannot explain impaired prefrontal function in conditions such as Alzheimer’s disease. Acetylcholine may work in tandem with other neuromodulators to affect prefrontal cortex function; alternatively, it may only be involved in very specific aspects of cortical function, for example representational plasticity.

Related Products: ME20.4-SAP (Cat. #IT-15)

Tai S, Ma J, Leung L (2007) Vestibular activation stimulates cholinergic system in the hippocampus. Neuroscience 2007 Abstracts 399.21/OO13. Society for Neuroscience, San Diego, CA.

Summary: The vestibular system has been suggested to participate in spatial navigation, a function ascribed to the hippocampus. We examined the mechanisms that induced hippocampal theta, a 4-10 Hz rhythm in the electroencephalogram (EEG), during vestibular activation in rats. Freely behaving rats were rotated at various speeds, on a vertical axis, in the light or dark. Hippocampal EEGs were recorded by implanted electrodes in hippocampal CA1, and analyzed by spectral analysis. A clear hippocampal theta rhythm was induced during immobility by rotations at different speeds (20-70 rpm). The rotation-induced theta was abolished, in light and dark settings, by muscarinic cholinergic receptor antagonist atropine sulfate (50 mg/kg i.p.) but not by atropine methyl nitrate (50 mg/kg i.p.), which did not pass the blood-brain barrier. Rotation-induced theta was attenuated in rats in which the cholinergic neurons in the medial septum (MS) were lesioned by 192 IgG-saporin (0.14 µg/0.4 µl infused bilaterally into the MS 10-20 days prior to the experiments). Cholinergic lesion in the MS was confirmed by a depletion of MS neurons that stained positively for choline acetyltransferase and an absence of acetylcholinesterase histochemical staining in the hippocampus. Bilateral lesion of the vestibular receptors (by 0.1 ml intratympanic injection of 300 mg/ml sodium arsanilate) also attenuated the rotation-induced theta rhythm. Vestibular lesion was confirmed by the contact righting test where lesioned rats will “walk” upside down on a Plexiglas sheet placed in contact with the soles of the feet while intact rats will right themselves immediately. In summary, an atropine-sensitive hippocampal theta is activated by septohippocampal cholinergic neurons which are in turn activated by vestibular stimulation. Vestibular-activated septohippocampal cholinergic activity is likely an important component of spatial navigation.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Burk JA, Altemose KE, Lowder MW (2007) Effects of spatial cueing on visual discrimination performance of rats following loss of basal forebrain corticopetal cholinergic neurons. Neuroscience 2007 Abstracts 424.4/HHH3. Society for Neuroscience, San Diego, CA.

Summary: Basal forebrain corticopetal cholinergic neurons are necessary for normal attentional performance. However, the exact attentional task parameters that are sufficient for inducing deficits following loss of basal forebrain corticopetal cholinergic neurons remain unclear. In Experiment 1, rats were trained to perform a spatially cued visual discrimination task (press a lever under an illuminated panel light) with explicit attentional demands removed. Animals then received infusions of the cholinergic immunotoxin, 192IgG-saporin, or saline into the basal forebrain. All animals were then tested in the same task trained before surgery and then task parameters, including the duration of visual signals and the inter-trial interval, were manipulated in order to tax attentional processing. Lesioned animals exhibited an initial increase in response latencies immediately following surgery but this deficit was not observed when task demands were increased. When the task was modified to remove spatial cueing, by presenting visual signals or no signal from a centrally-located panel light, lesioned animals exhibited an increase in lever press latencies compared to sham-lesioned animals. In Experiment 2, rats were trained in a visual discrimination task that, within each session, had blocks of trials with or without spatial cueing, using procedures similar to Experiment 1. After receiving intra-basalis infusions of 192IgG-saporin or saline, animals were tested for 12 sessions in the same task trained before surgery followed by one session in which the inter-trial interval was decreased. Lesioned animals did not exhibit deficits immediately following surgery, but did show elevated lever press latencies compared to sham-lesioned animals when the inter-trial interval was decreased. There was a trend for this lesioned-induced deficit to be more pronounced when spatial cues were not present. These experiments indicate that cortical acetylcholine is critical for maintaining normal visual discrimination performance when spatial cueing is not available. We speculate that, under conditions in which spatial cueing is unavailable, the lesion-induced increased lever press latencies reflect a disruption in recalling rules for an appropriate response.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Butt AE, Amodeo D, Flesher M, Marsa K, Holt R, Lladones R, Olney R, Haynes J, Kinney-Hurd BL, Dach N (2007) 192 IgG-saporin lesions of the cholinergic basal forebrain disrupt selective attention and trace conditioning but spare delay and long-delay conditioning. Neuroscience 2007 Abstracts 424.9/HHH8. Society for Neuroscience, San Diego, CA.

Summary: Recent research suggests that Pavlovian trace conditioning, but not delay conditioning, is a form of declarative memory that requires attention, where both of these processes depend on specific brain systems. For example, past research has shown that although amnesiac humans with damage to the hippocampus (HPC) acquire a normal conditioned response (CR) in delay conditioning paradigms, where the conditioned stimulus (CS) and unconditioned stimulus (US) partly overlap, they fail to acquire the CR in trace conditioning paradigms, where the CS and US are separated in time. Others have shown that the anterior cingulate cortex (ACC) in rats and the medial prefrontal cortex (mPFC) in rabbits are similarly necessary for trace but not delay conditioning. The basal forebrain cholinergic system (BFCS) has projections to the mPFC, ACC, and HPC. Given that each of these regions is critical for trace but not delay conditioning, we hypothesized that lesions of the BFCS using 192 IgG-saporin (SAP) would selectively impair trace but not delay or long-delay appetitive conditioning in rats. Additionally, given evidence suggesting BFCS involvement in attention, it was hypothesized that the addition of varying levels of distraction during the trace conditioning task would cause progressively greater degrees of impairment in the BFCS lesion groups compared to controls. In contrast, neither the control groups nor the BFCS lesion groups were expected be negatively affected by the addition of a distracting stimulus in the delay and long-delay conditioning tasks. Rats received bilateral SAP lesions or sham lesions of the BFCS prior to conditioning with a white noise CS and sucrose pellet US in either a delay, long-delay, or 10 s trace conditioning paradigm. Separate groups of BFCS lesion and control rats were subjected to varying levels of visual distraction (flashing house light) in each paradigm; no distraction, low distraction (continuously blinking light), and high distraction (intermittent, unpredictable flashing light). Results supported our hypotheses, with the BFCS lesion groups showing normal delay and long-delay conditioning regardless of level of distraction. In contrast, trace conditioning was impaired in the BFCS lesion groups, with progressively greater degrees of impairment occurring with greater levels of distraction, and complete disruption of learning in the high distraction condition. Together, these experiments suggest that the BFCS is necessary for normal trace conditioning, and that the BFCS is critically involved in selective attention tasks where animals must discriminate relevant stimuli from distracting background stimuli.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Likhtik E, Popa D, Apergis-Schoute J, Fidacaro GA, Pare D (2007) Lesion of intercalated (ITC) amygdala neurons interfere with extinction of classically conditioned fear responses. Neuroscience 2007 Abstracts 426.6/HHH29. Society for Neuroscience, San Diego, CA.

Summary: The acquisition of conditioned fear responses (CRs) is thought to involve the potentiation of synapses conveying information about the conditioned stimulus (CS) to the basolateral (BLA) amygdala. Expression of CRs would depend on the transfer of potentiated CS inputs by the BLA to the central amygdala (CE). In contrast, the mechanisms of extinction remain controversial. It has been proposed that ITC neurons, which receive BLA inputs and generate feedforward inhibition in CE, are in a key position to mediate extinction. In this view, NMDA-dependent potentiation of BLA inputs to ITC cells during extinction training, would dampen the impact of CS-related BLA activity on CE neurons, inhibiting CRs. However, this idea is difficult to test because ITC cells occur in small, lateromedially dispersed clusters, making conventional lesioning methods inadequate. Here, we took advantage of the fact that, compared to the rest of the amygdala, ITC cells express a much higher concentration of mu opioid receptors (muORs). As a result, we could lesion them by performing local injections of a peptide-toxin conjugate (demorphin conjugated to saporin, D-Sap) that selectively targets cells expressing muORs. Control rats received injections of saporin conjugated to a blank peptide (B-Sap). On Day 1, intact rats were subjected to a standard cued fear conditioning protocol in context A. On Day 2, they received 20 CS alone presentations in a different context (B). On Day 3, rats then received either D-Sap or B-Sap injections in the ITC cell masses. One week later, extinction recall was tested in context B with 10 CS alone presentations. Compared to control (B-Sap) rats (n=10), ITC-lesioned rats (n=5) had an extinction deficit (ANOVA, F=11.687, p = 0.005). Post-hoc t-tests comparing % time freezing during the first five or last five CSs revealed that rats with ITC lesions had significantly higher freezing levels throughout the extinction recall test (p<0.002 for both tests). These differences were not attributable to a non-specific increase in freezing or anxiety levels as exploratory behaviors in a novel open field in control and ITC-lesioned rats were indistinguishable. Overall, these results indicate that ITC cells are involved in the expression of extinction.

Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), Blank-SAP (Cat. #IT-21)

Stornetta RL, Takakura AC, Moreira TS, Mulkey DP, Bayliss DA, Guyenet PG (2007) Phox2b-expressing neurons of the retrotrapezoid nucleus (RTN) and central respiratory chemoreception in rats. Neuroscience 2007 Abstracts 230.9. Society for Neuroscience, San Diego, CA.

Summary: The RTN contains glutamatergic interneurons that are strongly activated by CO2 via acidification. These chemosensitive neurons are non-catecholaminergic and they express the transcription factor Phox2b. Although RTN chemoreceptors innervate selectively the brainstem regions that contain the respiratory rhythm and pattern generator (CPG), it is not yet clear whether these neurons drive inspiration or expiration, pump or airway muscles, autonomic circuits or all of the above. To determine whether RTN neurons drive inspiration, we examined whether their selective destruction modifies the CO2 sensitivity of the phrenic nerve discharge (PND) in anesthetized vagotomized rats. Using electrophysiological recordings in vivo and in slices, we found that the chemosensitive neurons of RTN express substance P receptors. We also found that these cells can be destroyed by local injection of a substance P agonist conjugated with saporin (SSP-SAP). The kill rate of RTN chemoreceptors was determined by counting the number of residual Phox2b-expressing non-catecholaminergic neurons present in this structure 15 days after toxin injection. Unilateral injection of 0.6 ng SSP-SAP destroyed 75% of the presumptive chemoreceptors on the injected side only. The lesion was selective because nearby neurons such as facial motoneurons, catecholaminergic and serotonergic cells were spared. SSP-SAP also spared the ventral respiratory column caudal to RTN except for a small amount of damage in the Bötzinger region closest to RTN. Unilateral lesion of the Phox2b-expressing neurons of RTN had no effect on PND and on respiratory chemoreception. However, in such rats, a single injection of the GABA-mimetic muscimol into the contralateral intact RTN instantly eliminated PND. After muscimol, PND did not usually reappear in the presence of hypercapnia up to 10% end-expiratory CO2. However, PND could typically be reactivated by strong stimulation of peripheral chemoreceptors which suggests that the respiratory oscillator had remained functional after muscimol. Unilateral injection of a lower dose of SSP-SAP (0.15 ng) had no effect on the Phox2b-expressing neurons of RTN. In such rats, unilateral injection of muscimol into the contralateral RTN had no detectable effect on PND and central chemoreception.

Related Products: SSP-SAP (Cat. #IT-11)